Journal: bioRxiv

Article Title: PIEZOs regulate oligodendrocyte sheath formation, expansion, and myelination potential

doi: 10.64898/2026.04.23.720488

Figure Lengend Snippet: A. Raw images from Fura2 calcium imaging after the application of DMSO, 30 µM Yoda1, and 200 mM KCl. Scale bar: 25 μm B. Representative calcium imaging trace from control ( Piezo1 Wt/Wt ;Olig2 Cre/Wt ) OPCs loaded with Fura2 and sequentially exposed to DMSO, 30 µM Yoda1, and 200 mM KCl. Trace averaged from 72 cells imaged from 1 coverslip. C. Representative calcium imaging trace from Piezo1 CKO cells ( Piezo1 Fl/Fl ;Olig2 Cre/Wt ) loaded with Fura2 and exposed to DMSO, 30 µM Yoda1, and 200 mM KCl. Trace averaged from 121 CKO cells from a single coverslip. D. Quantification of area under the 340/380 curve for each animal during application of Yoda1. The imaging data from 3 coverslips per animal were pooled for analysis for 3 animals per genotype. Data shown as mean ± SEM. p = 0.024 by student’s t-test, N = 3 animals per genotype, 971 control cells and 1043 cells from Piezo1 CKO cells, 3 independent isolations. E. Representative images from EdU proliferation assay of control or Piezo1 CKO cells. OLIG2 is a marker of OLCs, and EdU labels cells that divided over the 6.5-hour time course. F. Experimental design for the detection of proliferating cells in vitro . G. Quantification of the proportion of OLIG2+ cells labelled for EdU. N = 3 independent OPC isolations per genotype, with 818 cells assessed for controls and 947 cells for Piezo1 CKOs . p = 0.643 by paired t-test. H. Representative images from control or Piezo1 CKO cells fixed 48 hours into differentiation and stained for OLIG2, PDGFRA, and MBP. I. Quantification of the percentage of OLIG2+ cells that are PDGFRA+ OPCs. Control experimental totals: 859 cells, 3 animals, 3 independent OPC isolations CKO experimental totals: 824 cells, 3 animals, 3 independent OPC isolations. p = 0.207 by paired, 2-tailed t-test. J. Quantification of the percentage of OLIG2+ cells that are actively differentiating (PDGFRA-, MBP-). Control experimental totals: 859 cells, 3 animals, 3 independent OPC isolations CKO experimental totals: 824 cells, 3 animals, 3 independent OPC isolations. p = 0.101 by paired, 2-tailed t-test. K. Quantification of the percentage of OLIG2+ cells that are MBP+ OLs. Control experimental totals: 859 cells, 3 animals, 3 independent OPC isolations CKO experimental totals: 824 cells, 3 animals, 3 independent OPC isolations. p = 0.097 by paired, 2-tailed t-test. L. Representative images of a control and Piezo1 CKO cell plated onto nanofibers and allowed to differentiate for 7 days before being fixed and stained with DAPI and MBP. M. The number of sheaths per OL for Control and Piezo1 CKO cells. p = 0.767 by paired, 2-tailed, t-test. N. Average sheath length per OL for Control and Piezo1 CKO cells. p = 0.019 by paired, 2-tailed t-test. O. Average myelin output (summed sheath lengths per OL) for control ( Piezo1 wt/wt ;Olig2 Cre/wt ) and Piezo1 CKO cells. p = 0.032 by paired, 2-tailed t-test. For M-O, Control experimental totals: 50 cells, 3 animals, 3 independent cell isolations. CKO experimental totals: 48 cells, 3 animals, 3 independent cell isolations. For E, H, and L – Scale bar: 50 μm

Article Snippet: Piezo1 F/F mice were purchased from Jackson Laboratories (B6.Cg-Piezo1 tm2.1Apat/J; Jax strain: 029213) , and crossed to a Olig2-Cre (Olig2-TVA-IRES-Cre KIKO; Jax strain: 011103) mice which were a gift from Dr. David Rowitch.

Techniques: Imaging, Control, Proliferation Assay, Marker, In Vitro, Staining