The channel kinase, TRPM7, is required for early embryonic development

Abstract

Global disruption of transient receptor potential-melastatin-like 7 (Trpm7) in mice results in embryonic lethality before embryonic day 7. Using tamoxifen-inducible disruption of Trpm7 and multiple Cre recombinase lines, we show that Trpm7 deletion before and during organogenesis results in severe tissue-specific developmental defects. We find that Trpm7 is essential for kidney development from metanephric mesenchyme but not ureteric bud. Disruption of neural crest Trpm7 at early stages results in loss of pigment cells and dorsal root ganglion neurons. In contrast, late disruption of brain-specific Trpm7 after embryonic day 10.5 does not alter normal brain development. We developed induced pluripotent stem cells and neural stem (NS) cells in which Trpm7 disruption could be induced. Trpm7(-/-) NS cells retained the capacities of self-renewal and differentiation into neurons and astrocytes. During in vitro differentiation of induced pluripotent stem cells to NS cells, Trpm7 disruption prevents the formation of the NS cell monolayer. The in vivo and in vitro results demonstrate a temporal requirement for the Trpm7 channel kinase during embryogenesis.

Fig. 1.

TM-induced and tissue-specific Trpm7 disruption reveals a spatiotemporal requirement of the channel kinase for embryogenesis. (A) Trpm7 is temporally required for embryogenesis. TM-induced global Trpm7 disruption at E7–E9, but not at later stages (>E14.5 or adults), was consistently lethal. After timed breeding [male Trpm7^fl/fl (Cre-ER) mice with female Trpm7^fl/fl mice], TM was injected i.p. into pregnant females at E7, E7.5, E8.5, and E14.5. TM was also injected into 6-wk-old Trpm7^fl/fl (Cre-ER) adult mice to induce Trpm7 disruption. (B) Representative images of grossly deformed, nonviable embryos at E9.5 (Upper) and E10.5 (Lower). Trpm7 disruption was induced at E7–E9. Detailed results are described in Fig. S1. (C) Real-time PCR genotyping of genomic DNA from Trpm7^fl/fl [Nestin-Cre (ko)] and Trpm7^fl/fl littermate control (wt) mice indicates depletion of the fl allele (the loxP-flanked exon 17) in ko brain but not heart. Quantification of the fl level in ko mice was normalized to wt. (D) Depletion of TRPM7 protein in the ko brain. Brain lysates were prepared from wt, het, and ko mice and analyzed by Western blotting. The arrowhead indicates TRPM7. (E) Normal brain histology of Nestin-Cre Trpm7 ko mice. Representative H&E-stained hippocampal sections from 12-wk-old wt and ko mice. (F) Diagram showing the differential expression pattern of the HoxB7-Cre and Pax3-Cre transgenes at E11.5: Hox B7-Cre was expressed in the UB, whereas Pax-3 Cre was expressed in the MM. (G) Western blot analysis of TRPM7 from kidney lysates in wt and HoxB7-Cre sections from P12 or Pax-3 Cre Trpm7 ko mice. (H) Images of P12 kidneys (wt vs. ko) mediated by Pax3-Cre (Upper) and HoxB7-Cre (Lower). (I) H&E staining of sections from P12 kidneys (wt vs. ko). The HoxB7-Cre ko kidney histology was normal, whereas the Pax3-Cre ko kidney contained cysts and a reduced number of glomeruli.

Fig. 2.

Pax3-Cre–mediated Trpm7 disruption in kidney resulted in early defective nephrogenesis and a progressive kidney failure phenotype. (A) H&E staining of kidney sections at E14.5, E18.5, and P2 from Pax3-Cre Trpm7 ko mice. Spherical renal vesicles were present in E14.5 ko embryonic kidney, but comma- and S-shaped bodies were clearly absent (compared with wt control; arrows). There were reduced glomeruli in ko embryonic kidney at E18.5 and dilated tubules in the renal cortex of ko kidney at P2. (B) High-magnification images of dilated renal tubules in P2 ko kidney. (Left) Note even distribution of eosinophilic acellular material the tubular lumen of ko (example circled). (Right) Note well-organized basement membrane in wt kidney (arrow), and damaged ones in ko kidney (diffuse purple in periodic acid–Schiff stain). (C) Proximal tubular cysts first appeared at P4 in ko kidney. The cysts were associated with proximal tubules (AQP1⁺) but not connecting tubules (D28K⁺), distal convoluted tubules (NCC⁺), or collecting ducts (AQP2⁺). Cysts are highlighted by white dots. Nuclei were stained by DAPI.

Fig. 3.

Trpm7 is temporally required for development of NC-derived pigment cells and DRG sensory neurons. (A) Diagram showing unique pattern of Pax3-Cre expression in E10.5 NC. The Cre transgene does not faithfully follow the endogenous Pax3 expression pattern and was not expressed in the upper (cervical and part of the thoracic) region of the NC (18). (B) Unique phenotype of Pax-3 Cre Trpm7 ko mice (P2 or P12): loss of pigment only in the lower trunk and paresis of only the hind legs. (C) Absence of pigment cells only in the lower trunk of the ko mice. (Left) H&E (H.E.) staining shows that pigment cells are present in the upper but not lower trunk in dorsal skin from a P12 ko mouse. (Right) Toluidine blue (T.B.) staining of P2 dorsal lumbar skin sections shows loss of pigment cells in the lower trunk of the ko mouse compared with wt littermate controls. (D) Immunohistochemistry of P2 dorsal lumbar skin sections confirmed the absence of microphthalmia-associated transcription factor (MiTF⁺) (Left) or DCT⁺ (Right) pigment cells in the lower trunk of ko mice. (E) Merged immunofluorescence and differential interference contrast (DIC) images show that Trpm7 was disrupted in both the upper and lower trunk of a P2 ko mouse, but the lower trunk had few DCT⁺ pigment cells and lacked pigment in comparison to the upper trunk. (F) (Upper) H&E staining of E18.5 lumbar spinal cord and DRG. (Lower) Loss of TRPM7 in large-diameter dorsal root ganglion (DRG) sensory neurons in ko mice (immunofluorescence, P2 lumbar DRG).

Fig. 4.

Trpm7^fl/fl (Cre-ER) iPS cells and NS cells. (A) (Top) Isolated MEFs from E13.5 Trpm7^fl/fl (Cre-ER) embryos. iPS cells were generated by reprogramming Trpm7^fl/fl (Cre-ER) MEFs via retroviral expression of Oct4, Sox2, and Klf4. (Middle) ES cell-like morphology of iPS cells; phase contrast microscopy. (Bottom) Oct4 expression in iPS cells. (B) In vitro differentiation of iPS3 cells into ectodermal (Nestin⁺), endodermal (cytokeratin, Endo-A⁺), and mesodermal [PECAM1⁺, Troponin T⁺ (TnT⁺)] cells. (C) Real-time RT-PCR analysis of a panel of genes in two Trpm7^fl/fl (Cre-ER) iPS clones (IPS3 and iPS6). Both clones exhibit a similar expression pattern as J1 ES cells. Expression was normalized to that of J1 ES cells. (D) Five weeks after injection of Trpm7^fl/fl (Cre-ER) iPS3 in immunocompromised mice, all three germ layers were present in teratomas: neural epithelium (n; ectodermal), muscle and cartilage (m and c; mesodermal), and respiratory ducts (r; endodermal). Analysis of Trpm7^fl/fl (Cre-ER) iPS6 is shown in Fig. S2. (E) Trpm7^fl/fl (Cre-ER) NS cells were derived from pluripotent stem cells (29) (Materials and Methods). Monolayer of Trpm7^fl/fl (Cre-ER) NS cells (Middle) is a nearly a homogeneous population of NS cells, as shown by immunofluorescence staining with the anti-Nestin antibody (red; Bottom). Nuclei were stained by DAPI (blue; Bottom).

Fig. 5.

Trpm7^−/− NS cells survive, renew, and have normal total magnesium content. (A) Genomic DNA real-time PCR shows that 2-μM TM treatment of Trpm7^fl/fl (Cre-ER) NS cells for two passages (P1, P2) led to complete deletion of the fl allele, in comparison to untreated NS cells (P0), resulting in Trpm7^−/− NS cells (ko). (B) Whole-cell patch-clamp of ko cells confirms the loss of TRPM7 current. (Left) Representative whole-cell currents. (Right) Pooled results of TRPM7 current density at +100 mV in wt (n = 8) and ko (n = 8) NS cells. (C) Measurement of total Mg²⁺ content in wt and ko NS cells. Total Mg²⁺ and K⁺ were measured via ICP-MS from 4 wt and 3 ko NS cell samples; [Mg²⁺] was calculated based on the Mg²⁺/K⁺ ratio. (D) Proliferation assay (MTS) shows no significant difference between wt and ko NS cells at either day 1 or day 3. Additionally, there is similar proliferation in Trpm7^−/− and wt NS cells in the presence or absence of extracellular Mg²⁺ (10 mM).

Fig. 6.

Trpm7^−/− NS cells retain the potential to differentiate into neurons and astrocytes. Trpm7^fl/fl (wt) or Trpm7^−/− (ko) NS cells were seeded on chamber slides and then switched to differentiation medium for neurons (A) or astrocytes (B), as described in Materials and Methods. At days 0, 1, and 7, cells were fixed and double-stained with antibodies for Nestin or MAP2 (for neuron differentiation) or Nestin and GFAP (for astrocyte differentiation). Nuclei were counterstained with DAPI.

Fig. 7.

Cell death after Trpm7 disruption in iPS cells. (A) TM induced rapid Trpm7 disruption in Trpm7^fl/fl (Cre-ER) iPS cells. J1 ES cells and two iPS clones, iPS3 and iPS6 cells, were treated with 2 μM TM. (B) Of Trpm7^fl/fl (Cre-ER) cells treated with TM, only a few individual iPS3 and iPS6 cells survived. MgCl₂ (10 mM) in the culture medium did not rescue cells. J1 ES cells and controls were analyzed side-by-side. (C) TM-induced Trpm7 disruption in iPS cells resulted in death at 72 h. J1 ES and iPS 3 cells, before and after 72 h of TM treatment, were labeled with AxV-FITC and PI for flow cytometry analysis. Double-negative cells were viable (Group I), FITC⁺ PI⁻ cells (Group II) are early apoptotic cells, and late apoptotic/necrotic cells can be separated into two distinct groups: FITC⁺ PI^lo cells (Group III) and FITC⁺ PI^hi cells (group IV). *Necrosis that is common for adherent cells treated with trypsin (analysis with Flowjo software). (D) Progressive cell death was triggered by Trpm7 disruption. iPS6 cells were treated with 2 μM TM for 0, 24, 48, and 72 h, respectively, and double-stained with AxV-FITC and PI for flow cytometry analysis. Dynamic distribution of the four subgroups of cell populations during the time course indicates progressive cell death induced by Trpm7 disruption.

Fig. P1.

(A) TRPM7 is both an ion channel and a kinase that is temporally required for embryogenesis or embryo development. Most remarkably for an ion channel, one terminus of TRPM7 contains a kinase enzyme. The kinase is structurally homologous to other protein kinases, such as protein kinase A, but binds a metal ion (most likely Zn²⁺) (2). The drawing is fanciful because there is currently no high-resolution structure of a TRP channel transmembrane region. (B) Adult mice show the consequences of time-dependent deletion of the Trpm7 gene during development.