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Updated: Oct 2, 2026
Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes
Published on: February 13, 2021
Selective inhibition of murine palatal mesenchymal cell proliferation in vitro by secalonic acid D
Umesh M Hanumegowda1, Barbara M Judy, Wade V Welshons
1Department of Veterinary Biomedical Sciences, University of Missouri, 1600 E. Rollins, Columbia, Missouri 65211, USA.
Abstract:
Secalonic acid D (SAD), a teratogenic mycotoxin, induces cleft palate (CP) in the offspring of exposed mice by inhibiting palatal shelf growth. Since reduced proliferation, increased apoptosis, and/or decreased extracellular matrix (ECM) synthesis of palatal mesenchymal cells (PMC) can all contribute to smaller shelf size, the hypothesis that teratogenically relevant concentrations (0 to 120 microg/ml) of SAD will have adverse effects on one or more of these cellular processes was tested, using primary murine PMC cultures. Exposure to SAD resulted in significant and dose-dependent decreases in mesenchymal cell number, uptake of (3)H-thymidine, and expression of proliferating cell nuclear antigen (PCNA). Trypan blue dye exclusion assay, however, revealed significant cell death only at higher doses, suggesting that the decrease in cell number at lower (more realistic) doses is likely a consequence of reduced cell proliferation and not cell death. Further, negative results in the DNA fragmentation analysis following SAD exposure suggested that cell death caused by higher levels of SAD was unrelated to apoptosis. Similarly, results of (3)H-glucosamine uptake assay indicated inhibitory effect of SAD on accumulation of hyaluronic acid (HA) or sulfated glycosaminoglycans (sGAG) only at the highest dose tested. Also, SAD affected neither extracellular nor cell-associated fibronectin expression at any dose tested. Taken together, these data suggest that the pathogenesis of CP by SAD is likely a result of a reduction in the size of the palatal shelf caused by SAD-induced inhibition of mesenchymal cell proliferation.
Insights
Secalonic acid D (SAD) causes cleft palate in mice by inhibiting palatal shelf growth. SAD reduces mesenchymal cell proliferation, not cell death or extracellular matrix synthesis, leading to smaller palatal shelves.
Area of Science:
- Developmental toxicology
- Cell biology
- Teratology
Background:
- Secalonic acid D (SAD) is a mycotoxin known to cause birth defects.
- Cleft palate (CP) is a common congenital abnormality influenced by environmental factors.
- Palatal shelf growth is crucial for normal palate development and can be affected by reduced cell proliferation, increased apoptosis, or decreased extracellular matrix (ECM) synthesis.
Purpose of the Study:
- To investigate the effects of SAD on primary murine palatal mesenchymal cells (PMC).
- To determine if SAD inhibits cell proliferation, induces apoptosis, or affects ECM synthesis in PMCs at teratogenic concentrations.
- To elucidate the cellular mechanisms underlying SAD-induced cleft palate.
Main Methods:
- Primary murine PMC cultures were exposed to varying concentrations of SAD (0 to 120 microg/ml).
- Cell proliferation was assessed by measuring cell number, (3)H-thymidine uptake, and proliferating cell nuclear antigen (PCNA) expression.
- Cell death was evaluated using trypan blue dye exclusion and DNA fragmentation assays.
- ECM synthesis was analyzed by (3)H-glucosamine uptake for hyaluronic acid (HA) and sulfated glycosaminoglycans (sGAG), and fibronectin expression was assessed.
Main Results:
- SAD exposure caused significant, dose-dependent decreases in PMC number, (3)H-thymidine uptake, and PCNA expression.
- Significant cell death was observed only at higher SAD doses, and DNA fragmentation assays indicated it was not apoptotic.
- SAD had minimal impact on HA and sGAG accumulation and did not affect fibronectin expression at any tested dose.
Conclusions:
- SAD inhibits palatal mesenchymal cell proliferation in a dose-dependent manner.
- Reduced cell proliferation, rather than apoptosis or altered ECM synthesis, is the primary mechanism for SAD-induced inhibition of palatal shelf growth.
- These findings clarify the cellular pathogenesis of SAD-induced cleft palate, highlighting the critical role of proliferation inhibition.
