Knockdown of Mns1 Increases Susceptibility to Craniofacial Defects Following Gastrulation-Stage Alcohol Exposure in Mice

Abstract

Background: MNS1 (meiosis-specific nuclear structural protein 1) is necessary for motile cilia function, such as sperm flagella or those found in the embryonic primitive node. While little is known regarding the function or expression pattern of MNS1 in the embryo, co-immunoprecipitation experiments in sperm have determined that MNS1 interacts with ciliary proteins, which are also important during development. Establishment of morphogenic gradients is dependent on normal ciliary motion in the primitive node beginning during gastrulation (gestational day [GD] 7 in the mouse, second-third week of pregnancy in humans), a critical window for face, eye, and brain development and particularly susceptible to perturbations of developmental signals. The current study investigates the role of Mns1 in craniofacial defects associated with gastrulation-stage alcohol exposure.

Methods: On GD7, pregnant Mns1^+/- dams were administered 2 doses of ethanol (5.8 g/kg total) or vehicle 4 hours apart to target gastrulation. On GD17, fetuses were examined for ocular defects by scoring each eye on a scale from 1 to 7 (1 = normal, 2 to 7 = defects escalating in severity). Craniofacial and brain abnormalities were also assessed.

Results: Prenatal alcohol exposure (PAE) significantly increased the rate of defects in wild-type fetuses, as PAE fetuses had an incidence rate of 41.18% compared to a 10% incidence rate in controls. Furthermore, PAE interacted with genotype to significantly increase the defect rate and severity in Mns1^+/- (64.29%) and Mns1^-/- mice (92.31%). PAE Mns1^-/- fetuses with severe eye defects also presented with craniofacial dysmorphologies characteristic of fetal alcohol syndrome and midline tissue loss in the brain, palate, and nasal septum.

Conclusions: These data demonstrate that a partial or complete knockdown of Mns1 interacts with PAE to increase the susceptibility to ocular defects and correlating craniofacial and brain anomalies, likely though interaction of alcohol with motile cilia function. These results further our understanding of genetic risk factors that may underlie susceptibility to teratogenic exposures.

Keywords: Birth Defects; Development; Ethanol; Fetal Alcohol Syndrome; Transgenic Mice.

Figure 1.

Eye defect scoring system used in the current study. A score of 1 indicated a normal eye. Scores of 2 – 7 indicate abnormal eyes with the severity of the defect increasing with higher numbers. Defects include microphthalmia (small eye size), coloboma (abnormality of the iris or lens, seen in scores 4 and 5), and partial to complete anophthalmia (scores 6–7).

Figure 2.

Characterization of Mns1^−/− mice. In 67% of Mns1^−/− mice, situs inversus was observed. A) An example of a fetal heart from a vehicle-treated, WT fetus with the apex of the heart pointing towards the mouse’s left. B) In KO mice displaying situs inversus, the apex of the heart was pointing towards the animal’s right. C) Brain of an untreated adolescent WT mouse showing normal brain structure. This section is from similar bregma coordinates as the brain of an untreated adolescent KO mouse (D). This brain shows severe hydrocephalus that has created enlarged ventricles and compressed the cortical tissue. The hippocampus, visible at this bregma (~−1.34 mm) in the normal brain, is not visible in the brain with hydrocephalus as it emerges more posteriorly due to tissue compression.

Figure 3.

Incidence and severity of ocular defects following GD7 alcohol exposure. A) Incidence of ocular defects expressed as percent of total fetuses. The most severely affected eye for each fetus was counted for this graph. For the analysis, each fetus was scored as “affected” or “unaffected” based on the presence or absence of a defect in either eye. PAE significantly increased the incidence of ocular defects in WT fetuses (* = p < 0.05 PAE-WT vs. Veh-WT). Within the PAE group, alcohol interacted with genotype to significantly increase the rate of ocular defects per fetus (†† = p < 0.01, PAE-KO vs. PAE-WT). Heterozygous mice did not significantly differ from either KO or WT. Genotype did not affect the rate of spontaneous eye defects in the vehicle-treated group. B) Incidence of ocular defects expressed as the percent of total eyes (two per fetus). These data demonstrate that for some affected fetuses, only one eye was malformed. Severity of eye defects determined that the total number of “non-subtle” eye defects was significantly greater in PAE WT fetuses compared to vehicle-treated WT (* = p < 0.05 PAE-WT vs. Veh-WT). Within the PAE group, alcohol interacted with genotype to significantly increase the rate of non-subtle eye defects († = p < 0.05, PAE-KO vs. PAE-WT; ^ = p < 0.05, PAE-KO vs. PAE-Het). Genotype did not affect the eye defect severity in the vehicle-treated group. For panels A and B, n’s for each group: Veh-WT: 20, Veh-Het: 32, Veh-KO: 8, PAE-WT: 17, PAE-Het: 28; PAE-KO: 13. C) The eye of a vehicle-treated fetus (score: 1). Following GD7 alcohol exposure, severe eye defects were observed in both Mns1^+/− and Mns1^−/− fetuses, including microphthalmia, the presence of colobomas (D; score: 4), and partial anophthalmia (E; score: 6).

Figure 4.

Craniofacial and CNS defects (black arrows) following GD7 alcohol exposure in Mns1^+/− and Mns1^−/− mice. A, D, H) Examples of the face (A), nasal cavity and palate (D), and brain (H) of vehicle-treated WT fetuses. B – C) Two PAE fetuses displaying a thin upper lip and smooth philtrum similar to the classic facial dysmorphologies associated with FAS. E) Nasal cavity and septum defects in a PAE fetus. F-G) Cleft palates and improper fusion of the nasal septum to the palatal shelf in PAE fetuses. I) Holoprosencephaly and J) dysgenesis of midline septal tissue in the PAE fetal brain.