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, 18 (22), 4367-75

Mice Defective in Trpm6 Show Embryonic Mortality and Neural Tube Defects

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Mice Defective in Trpm6 Show Embryonic Mortality and Neural Tube Defects

Roxanne Y Walder et al. Hum Mol Genet.

Abstract

The syndrome of hypomagnesemia with secondary hypocalcemia is caused by defective TRPM6. This protein is an ion channel that also contains a kinase in its C-terminus. It is usually diagnosed in childhood and, without treatment with supplemental Mg, affected children suffer from mental retardation, seizures and retarded development. We developed a mouse lacking Trpm6 in order to understand in greater detail the function of this protein. In contrast to our expectations, Trpm6(-/-) mice almost never survived to weaning. Many mice died by embryonic day 12.5. Most that survived to term had neural tube defects consisting of both exencephaly and spina bifida occulta, an unusual combination. Feeding dams a high Mg diet marginally improved offspring survival to weaning. The few Trpm6(-/-) mice that survived were fertile but matings between Trpm6(-/-) mice produced no viable pregnancies. Trpm6(+/-) mice had normal electrolytes except for modestly low plasma [Mg]. In addition, some Trpm6(+/-) mice died prematurely. Absence of Trpm6 produces an apparently different phenotype in mice than in humans. The presence of neural tube defects identifies a previously unsuspected role of Trpm6 in effecting neural tube closure. This genetic defect produces one of very few mouse models of spina bifida occulta. These results point to a critical role of Trpm6 in development and suggest an important role in neural tube closure.

Figures

Figure 1.
Figure 1.
(A) Litter of 12.5 day embryos identified by genotype. Parents were Trpm6+/−. The one Trpm6−/− (KO) embryo appears smaller than the others. (B) Weight of Trpm6 embryos at 16.5–18.5 dpc. N = 45 (+/+), 39 (+/−), 29 (−/−). *P < 0.05 by ANOVA. Difference in weight was evident for each litter.
Figure 2.
Figure 2.
(A) Example of Trpm6−/− newborn displaying exencephaly (arrows). Left photograph is the mouse at birth. Right shows a sagittal section stained with alcian blue. In addition to exencephaly, the mouse has a short snout and poorly developed facial bones (arrow with dot). Spina bifida occulta shown by arrows with dashed lines. (B) Example of defect in spinal closure in lower spine (arrows—dashed lines) in an 18.5 day embryo. This mouse also has defective brain and facial development.
Figure 3.
Figure 3.
Northern blot of Trpm6 expression in normal mouse embryos. The arrow indicates the position of the full size Trpm6 message. Lane numbers represent days post conception (dpc). Samples from embryonic day 4.5–6.5 include extra-embryonic tissues and maternal uterus, whereas samples from days 7.5 to 9.5 are conceptuses, including embryo and extra-embryonic tissue. Samples from days 10.5 to 18.5 contain only embryonic tissue. Mouse Trpm6 mRNA shows a distinct peak at 10.5 days. There is also an abundant signal at 4.5 dpc, which may be from maternal tissue.
Figure 4.
Figure 4.
Plasma [Mg] and response to low Mg diet in Trpm6+/+, and Trpm6+/−, mice. (A) Plasma [Mg] by genotype and gender on a normal diet. Open bars, Trpm6+/+; hatched bars, Trpm6+/−. Both gender and genotype were significantly different by two ways ANOVA; there was no interaction. Nine to 14 mice in each group. *P < 0.05 compared with Trpm6+/+. (BD) Response to 5 days of a low Mg diet. (B) Plasma [Mg] before (open bars, n = 4,6) and after (hatched bars, n = 4,5) low Mg diet. (C) Urinary Mg excretion in Trpm6+/+ (circles) and Trpm6+/− (squares) mice during the low Mg diet. There was no difference in plasma [Mg] after the low Mg diet by genotype or gender. There was no difference in fecal or urinary excretory response to low Mg diet by genotype or gender at any day.
Figure 5.
Figure 5.
(A) Targeting strategy for deletion of Trpm6. Upon homologous recombination, the 3′ end of exon 5 and all of exons 6 and 7 are replaced with a neomycin cassette. (B) PCR genotyping of Trpm6 mouse DNA. Primers were designed to amplify the wild-type allele (lanes A and B) and the targeted allele (lanes C and D). Sample 1: Trpm6+/+; Sample 2: Trpm6+/−; Sample 3: Trpm6−/− DNA.

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