Skip to main content
. 2022 Apr 6;11:e68598. doi: 10.7554/eLife.68598

Figure 6. Loss of mesenchymal Tfam or Rhot1 compromises fibroblast/myofibroblast migration.

(A) Immunostaining of lungs collected from control and Tfamf/f; PdgfraCre/+ mice at postnatal (P) day 2 and 5, some of which were injected with EdU as indicated. (B) Quantification of fibroblast/myofibroblast proliferation in control and Tfamf/f; PdgfraCre/+ lungs at P2, P3, and P5 (n = 3 for each group). The rate of fibroblast/myofibroblast proliferation was calculated as the ratio of the number of EdU+ fibroblasts/myofibroblasts (EdU+ PDGFRA+) to the number of fibroblast/myofibroblasts (PDGFRA+). The percentage of proliferating fibroblasts/myofibroblasts was reduced in Tfamf/f; PdgfraCre/+ compared to controls at P3 and P5. (C) qPCR analysis of gene expression in control and Tfamf/f; PdgfraCre/+ lungs at P3 (n = 3 for each group). The expression levels of Pdgfra, Acta2, and Eln were significantly reduced in Tfamf/f; PdgfraCre/+ lungs compared to controls. (D) Wound recovery assays to assess the migratory ability of fibroblasts/myofibroblasts derived from control and Tfamf/f; PdgfraCre/+ lungs (n = 3 for each group). Within 36–48 hr, the wound area has been populated by migrating fibroblasts/myofibroblasts derived from control lungs. By contrast, fewer fibroblasts/myofibroblasts from Tfamf/f; PdgfraCre/+ lungs reached the wound area within the same time frame. Wound recovery by fibroblasts/myofibroblasts from control and Tfamf/f; PdgfraCre/+ lungs was quantified. (E) Immunostaining of lungs collected from control and Rhot1f/f; PdgfraCre/+ mice at P5 and P10, some of which were injected with EdU as indicated. (F) Quantification of fibroblasts/myofibroblasts proliferation in control and Rhot1f/f; PdgfraCre/+ lungs at P5 and P10 (n = 3 for each group). The percentage of proliferating fibroblasts/myofibroblasts was decreased in Rhot1f/f; PdgfraCre/+ compared to controls at P10. (G) qPCR analysis of gene expression in control and Rhot1f/f; PdgfraCre/+ lungs at P7 (n = 3 for each group). The expression levels of Eln were significantly reduced in Rhot1f/f; PdgfraCre/+ lungs in comparison with controls. (H) Wound recovery assays to assess the migratory ability of fibroblasts/myofibroblasts derived from control and Rhot1f/f; PdgfraCre/+ lungs (n = 3 for each group). Fewer fibroblasts/myofibroblasts from Rhot1f/f; PdgfraCre/+ lungs reached the wound area within the same time frame compared to controls. Wound recovery by fibroblasts/myofibroblasts from control and Rhot1f/f; PdgfraCre/+ lungs was quantified. All values are mean ± SEM. *p<0.05; ns, not significant (unpaired Student’s t-test).

Figure 6—source data 1. EdU quantification, relative transcript levels, and quantification of wound recovery.

Figure 6.

Figure 6—figure supplement 1. Tfam- but not Rhot1-deficient fibroblasts display short tubular and fragmented mitochondria.

Figure 6—figure supplement 1.

(A) Immunostaining of fibroblasts derived from control and Tfamf/f; PdgfraCre/+ lungs at postnatal (P) day 5. Cells were lentivirally transduced with Mito-7-mEmerald-expressing constructs to visualize mitochondria and stained with MTCO1 antibodies to detect cytochrome c oxidase. (B) Quantification of mitochondrial network and morphology. Tubular mitochondria were the dominant form in control fibroblasts, while short tubular and fragmented mitochondria were found in Tfam-deficient fibroblasts. (C) Immunostaining of fibroblasts derived from control and Rhot1f/f; PdgfraCre/+ lungs at P5. (D) Quantification of mitochondrial network and morphology. No apparent difference in mitochondrial morphology was found between control and Rhot1-deficient fibroblasts.
Figure 6—figure supplement 1—source data 1. Quantification of mitochondrial morphology.
Figure 6—figure supplement 2. The migratory defect in Tfam- and Rhot1-deficient fibroblasts/myofibroblasts is rescued by expression of TFAM and RHOT1, respectively.

Figure 6—figure supplement 2.

(A) Wound recovery assays to assess the migratory ability of fibroblasts/myofibroblasts derived from Tfamf/f; PdgfraCre/+ mouse lungs (n = 3 for each group). Cells were transduced with control or mouse TFAMFLAG (mTFAMFLAG)-expressing constructs. Within 36–48 hr, the wound area has been populated by migrating Tfam-deficient fibroblasts/myofibroblasts expressing mTFAMFLAG. By contrast, fewer Tfam-deficient fibroblasts/myofibroblasts reached the wound area within the same time frame. (B) Wound recovery by Tfam-deficient fibroblasts/myofibroblasts and Tfam-deficient fibroblasts/myofibroblasts expressing mTFAMFLAG was quantified. (C) Wound recovery assays to assess the migratory ability of fibroblasts/myofibroblasts derived from Rhot1f/f; PdgfraCre/+ mouse lungs (n = 3 for each group). Cells were transduced with control or mouse RHOT1FLAG (mRHOT1FLAG)-expressing constructs. Within 36–48 hr, the wound area has been populated by migrating Rhot1-deficient fibroblasts/myofibroblasts expressing mRHOT1FLAG. By contrast, fewer Rhot1-deficient fibroblasts/myofibroblasts reached the wound area within the same time frame. (D) Wound recovery by Rhot1-deficient fibroblasts and Rhot1-deficient fibroblasts/myofibroblasts expressing mRHOT1FLAG was quantified. (E) Western blotting of cell lysates from control fibroblasts/myofibroblasts, Tfam-deficient and Rhot1-deficient fibroblasts/myofibroblasts expressing mTFAMFLAG or mRHOT1FLAG, respectively, as indicated. The numbers indicate the positions of protein size standards.
Figure 6—figure supplement 2—source data 1. Quantification of wound recovery.