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Gestational exposure to ethanol suppresses msx2 expression in developing mouse embryos
L Rifas1, D A Towler, L V Avioli
1Department of Internal Medicine, Divisions of Bone and Mineral Diseases and Molecular Pharmacology, Washington University School of Medicine and Barnes-Jewish Hospital North, St. Louis, MO 63110, USA. lrifas@imgate.wustl.edu
Abstract:
Ethanol acts as a teratogen in developing fetuses causing abnormalities of the brain, heart, craniofacial bones, and limb skeletal elements. To assess whether some teratogenic actions of ethanol might occur via dysregulation of msx2 expression, we examined msx2 expression in developing mouse embryos exposed to ethanol on embryonic day (E) 8 of gestation and subjected to whole mount in situ hybridization on E11-11.5 using a riboprobe for mouse msx2. Control mice exhibited expression of msx2 in developing brain, the developing limb buds and apical ectodermal ridge, the lateral and nasal processes, olfactory pit, palatal shelf of the maxilla, the eye, the lens of the eye, otic vesicle, prevertebral bodies (notochord), and endocardial cushion. Embryos exposed to ethanol in utero were significantly smaller than their normal counterparts and did not exhibit expression of msx2 in any structures. Similarly, msx2 expression, as determined by reverse transcription-PCR and Northern blot hybridization, was reduced approximately 40-50% in fetal mouse calvarial osteoblastic cells exposed to 1% ethanol for 48 hr while alkaline phosphatase was increased by 2-fold and bone morphogenetic protein showed essentially no change. Transcriptional activity of the msx2 promoter was specifically suppressed by alcohol in MC3T3-E1 osteoblasts. Taken together, these data demonstrate that fetal alcohol exposure decreases msx2 expression, a known regulator of osteoblast and myoblast differentiation, and suggest that one of the "putative" mechanisms for fetal alcohol syndrome is the inhibition of msx2 expression during key developmental periods leading to developmental retardation, altered craniofacial morphogenesis, and cardiac defects.
Insights
Ethanol exposure in pregnant mice significantly reduced msx2 gene expression in developing embryos and fetal cells. This inhibition of msx2 may explain developmental abnormalities seen in fetal alcohol syndrome.
Area of Science:
- Developmental biology
- Teratology
- Molecular genetics
Background:
- Ethanol is a known teratogen causing fetal abnormalities.
- msx2 is a gene crucial for skeletal and craniofacial development.
Purpose of the Study:
- To investigate if ethanol-induced teratogenicity involves the dysregulation of msx2 gene expression.
- To examine the effect of ethanol on msx2 expression during embryonic development.
Main Methods:
- Exposure of pregnant mice to ethanol on embryonic day 8.
- Whole mount in situ hybridization to detect msx2 expression in E11-11.5 embryos.
- Reverse transcription-PCR and Northern blot analysis on fetal osteoblastic cells exposed to ethanol.
- Analysis of msx2 promoter activity in osteoblasts.
Main Results:
- Ethanol-exposed embryos were smaller and lacked msx2 expression in key developmental structures.
- msx2 expression was reduced by 40-50% in fetal osteoblastic cells treated with ethanol.
- Alkaline phosphatase activity increased twofold in ethanol-exposed osteoblasts.
- Ethanol suppressed msx2 promoter activity in osteoblasts.
Conclusions:
- Fetal alcohol exposure decreases msx2 expression, a regulator of osteoblast and myoblast differentiation.
- Inhibition of msx2 expression is a potential mechanism underlying developmental retardation, craniofacial defects, and cardiac abnormalities in fetal alcohol syndrome.