Deletion of Trpm7 disrupts embryonic development and thymopoiesis without altering Mg2+ homeostasis

Abstract

The gene transient receptor potential-melastatin-like 7 (Trpm7) encodes a protein that functions as an ion channel and a kinase. TRPM7 has been proposed to be required for cellular Mg2+ homeostasis in vertebrates. Deletion of mouse Trpm7 revealed that it is essential for embryonic development. Tissue-specific deletion of Trpm7 in the T cell lineage disrupted thymopoiesis, which led to a developmental block of thymocytes at the double-negative stage and a progressive depletion of thymic medullary cells. However, deletion of Trpm7 in T cells did not affect acute uptake of Mg2+ or the maintenance of total cellular Mg2+. Trpm7-deficient thymocytes exhibited dysregulated synthesis of many growth factors that are necessary for the differentiation and maintenance of thymic epithelial cells. The thymic medullary cells lost signal transducer and activator of transcription 3 activity, which accounts for their depletion when Trpm7 is disrupted in thymocytes.

Fig. 1. I_MIC and Mg2+ homeostasis in Trpm7-deficient cells

(A) I-V relationship of I_MIC in wt and Trpm7-deficient [knockout (KO)] thymocytes. (B) I_MIC densities in wt (n = 13 cells) and KO (n = 18 cells) T lymphocytes (P <0.0001, two samples independent t test). Box charts are shown as a box (25 to 75 percentile), vertical bars (5 to 95 percentile), and data point (diamonds) overlap with the mean value (empty square) and median value (horizontal line in the box). (C) I_MIC densities in wt (n = 9 cells) and KO (n = 10 cells) thymocytes (P = <0.0001, two samples independent t test). (D) Mg²⁺ uptake in wt T lymphocytes loaded with KMG104AM, as indicated by the averaged ratio of fluorescence intensity F at indicated time (seconds) over the initial fluorescence F0 at 0 s. Mg²⁺ uptake in the absence (blue, n =25 cells) or presence (red, n = 25 cells) of 0.5 mM 2-APB is shown. (E) ICP-MS quantitation of total Ca²⁺ and Mg²⁺ in HNO₃ extracts. Average concentration of total cellular Mg²⁺ (n = 3 mice) as calculated by normalizing to a [K⁺] of 120 mM is shown. Error bars indicate ± SD. (F) Mg²⁺ uptake in wt (blue, n = 117 cells) and KO (red, n =102 cells) thymocytes. [Ca²⁺] and [Mg²⁺] are in mM.

Fig. 2. Deletion of Trpm7 in thymocytes leads to defective thymopoiesis

(A) Hematoxylin and eosin–staining of thymus sections from 12-week-old wt (top) and KO (bottom) mice at 4× (left) and 20× (right) magnification (red box). The boundary between medullary and cortical regions is highlighted with a solid white line where evident. (B) Thymocytes were immunolocalized by antibody-to-CD3 staining (brown) against a nuclear counterstain (blue) in the thymus sections obtained from wt (top) and Trpm7-deficient (bottom) mice.; (Left): 4× magnification. Red boxes indicate the areas that are shown at 20× magnification to the right. The CD3+ T cell–enriched medullary regions are highlighted in a wt thymus (see fig. S6 for larger images). (C) Box chart showing the reduced number of thymocytes in Trpm7-deficient mice (red, n = 9 mice) as compared with wt mice (black, n = 9 mice). Box charts shown as a box (±SD), vertical bars (maximum-minimum values), and data overlap. The P values in all of the box charts were calculated using the two-sample independent t test. (D) Flow cytometry of CD4 and CD8 on thymocytes from wt and KO mice. (E) Box charts comparing the total number of thymocytes in the DN, DP, CD4+, and CD8+ thymocytes are shown (n = 7 mice).

Fig. 3. Trpm7-deficient thymocytes are partially blocked at the DN3 stage

(A) Flow cytometry of CD44 and CD25 expression in DN (CD4− CD8−) thymocytes. Thymocytes were stained with antibody to CD4 [fluorescein isothiocyanate (FITC)], antibody to CD8 (FITC), antibody to CD44 [phycoerythrin (PE)] and antibody to CD25 [phosphatidylcholine 7 (PC7)]. FITC-negative cells (DN) were analyzed for CD44 and CD25 expression. (B) (Left) Overlay of cell-surface CD25 expression in wt (black) and Trpm7-deficient (red) thymocytes. (Right) Overlay of CD25 expression in DN thymocytes. (C) Box charts showing percentage of DN population found in the (left to right) DN1, DN2, DN3, and DN4 stages. (D) Box charts showing total number of thymocytes found in the (left to right) DN1, DN2, DN3, and DN4 stages.

Fig. 4. Deletion of Trpm7 in thymocytes results in progressive loss of medullary epithelial cells

(A) Immunofluoresence staining of thymus sections with antibodies to thymic epithelial markers K8 (red) and K5 (green). Letters indicate thymic cortex (C) and medullary (M) regions. Scale bar, 200 μm. (B) Staining of thymus sections to CD3 (green) and K8 (red). (C) Dysregulated mRNA encoding growth factors in knockout thymocytes relative to wt thymocytes identified by quantitative RT-PCR. Growth factors with increased (blue) or decreased (red) mRNA abundance are presented as average ΔΔCt values (n = 3 mice). Error bars indicate SD. A complete list of quantitative RT-PCR results is in fig. S9. (D) Immunofluoresence staining of thymus sections with antibody to STAT3 (red) and K5 (green). (E) Immunofluoresence staining of phospho-STAT3 (Tyr705) (red) and K5 (green) in thymus sections. Scale bar, 20 μm.