Abstract
In humans, germline mutations in Trpm6 cause autosomal dominant hypomagnesemia with secondary hypocalcemia disorder. Loss of Trpm6 in mice also perturbs cellular magnesium homeostasis but additionally results in early embryonic lethality and neural tube closure defects. To define the mechanisms by which TRPM6 influences neural tube closure, we functionally characterized the role of TRPM6 during early embryogenesis in Xenopus laevis. The expression of Xenopus TRPM6 (XTRPM6) is elevated at the onset of gastrulation and is concentrated in the lateral mesoderm and ectoderm at the neurula stage. Loss of XTRPM6 produced gastrulation and neural tube closure defects. Unlike XTRPM6′s close homologue XTRPM7, whose loss interferes with mediolateral intercalation, depletion of XTRPM6 but not XTRPM7 disrupted radial intercalation cell movements. A zinc-influx assay demonstrated that TRPM6 has the potential to constitute functional channels in the absence of TRPM7. The results of our study indicate that XTRPM6 regulates radial intercalation with little or no contribution from XTRPM7 in the region lateral to the neural plate, whereas XTRPM7 is mainly involved in regulating mediolateral intercalation in the medial region of the neural plate. We conclude that both TRPM6 and TRPM7 channels function cooperatively but have distinct and essential roles during neural tube closure.
Highlights
The expected Mendelian ratio, suggesting that the embryonic mortality of Trpm6-deficient mice was mainly due to the loss of TRPM6 activity in placental cells
Unlike Xenopus laevis TRPM7 (XTRPM7), which at the neurula stage, shows high expression in the brain and neural plate, Xenopus TRPM6 (XTRPM6) was weakly expressed in the neural plate, and high level of XTRPM6 RNA was detected in the lateral and anterior regions (Fig. 1b, stage 17)
Supplementation of the medium with excess Mg2+ was more effective in rescuing the gastrulation defects caused by the XTRPM6-morpholino oligonucleotide (MO) in a dose-dependent manner (Supplementary Figure 3), suggesting that similar to what we previously reported for the XTRPM7 MO, the gastrulation defects produced by the XTRPM6 MO is dependent, Figure 1
Summary
The expected Mendelian ratio, suggesting that the embryonic mortality of Trpm6-deficient mice was mainly due to the loss of TRPM6 activity in placental cells. Depletion of TRPM7 from developing Xenopus laevis embryos using anti-sense morpholino technology produced defective gastrulation phenotypes, consistent with the phenotype documented in mice[16]. In Xenopus laevis TRPM7 (XTRPM7) depleted embryos, axial extension was impaired, resulting in a severe dorsal-flexure and failure of the neural tube to close. As the phenotypes caused by depletion of TRPM7 in Xenopus could be suppressed by Mg2+ supplementation as well as by expression of the Mg2+ transporter SLC41A2, our studies suggested a requirement for Mg2+ for gastrulation. The phenotypes caused by depletion of XTRPM7 could be rescued by expression of exogenous TRPM6, suggesting that TRPM6 could functionally compensate for TRPM7 during early embryogenesis[17,18]. We report that TRPM6 functions independently of TRPM7 to influence radial cell intercalation, playing a unique but complementary role to TRPM7 for successful completion of gastrulation and neural tube closure
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