Fig 2: scRNAseq reveals regional heterogeneity among adult neural stem cells.(A-C) UMAP plots of Nkx2.1 (A), Gsx2 (B), and Emx1 (C) expression in the scRNA-Seq neurogenic lineage (see also Figure 3—figure supplement 1). (D) Schematic of the region identity prediction calculation, where anchor gene sets (dashed gray lines) are calculated between Ventral Dissection (magentas) and Dorsal Dissection (greens) sNucRNA-Seq B cell nuclei and scRNAseq B cells (blues) and each scRNAseq B cell is given a Dorsal or Ventral Dissection predicted identity score (see also Figure 3—figure supplement 2). (E) The net predicted identity scores of each scRNA-Seq B cell plotted in UMAP space, where strongly Dorsal Dissection predicted identity cells are dark green, and strongly Ventral Dissection predicted identities are dark magenta. Dark gray cells were not included in the analysis. (F) Dot plot of the average Dorsal or Ventral Dissection predicted identity scores for scRNA-Seq B cell clusters B(14) and B(5+22). (G) UMAP plot of B cell cluster identities used in the following analysis: B(14) (light blue) and B(5+22) (dark blue). (H) (i) Venn diagram summarizing differential gene expression analysis between clusters B(14) (light blue) and B(5+22) (dark blue). (ii) Numbers of candidate marker genes identified after selecting significantly upregulated genes expressed in no more than 40% of cells of the other cluster. (I) Heatmap depicting expression of the top 10 differentially expressed genes between clusters B(14) (left) and B(5+22) (right). (J) UMAP plot of Dio2 expression in the scRNA-Seq neurogenic lineage. (K - L) Confocal micrographs of Dio2 RNA (magenta), S100b (green), and GFAP (white) protein expression in the dorsal (K) and ventral (L) V-SVZ. (M-N). Quantification of DAPI+, S100ß- Dio2 RNAscope puncta along the length of the V-SVZ (0 = ventral-most extent, 1 = dorso-lateral-most extent of the wedge) with the median puncta distribution location plotted as a horizontal line for each sample (n=3, each indicated by a different shade, ~ bregma 1.18, 0.98 and 0.62 mm). N. Summary schematic of Dio2 expression in three rostro-caudal coronal sections (~bregma 1.50, 0.98 and 0.14 mm); strong expression (magenta); sparse (light magenta). Boxed areas denote locations of dorsal (K) and ventral (L) high-magnification images. (O) UMAP plot of Urah expression in the scRNA-Seq neurogenic lineage. (P - Q) Confocal micrographs of Urah RNA (magenta), S100b (green), and GFAP (white) protein expression in the dorsal (P) and ventral (Q) V-SVZ. (R-S) Quantification of DAPI+, S100ß- Urah RNAscope puncta along the length of the V-SVZ (0 = ventral-most extent, 1 = dorso-lateral-most extent of the wedge) with the median puncta distribution location plotted as a horizontal line for each sample (n=3, each indicated by a different shade, bregma~ 1.34, 1.18 and 0.98 mm). S. Summary schematic of Urah expression in three rostro-caudal coronal sections (~bregma 1.50, 0.98 and 0.14 mm); strong expression (magenta); sparse (light magenta). Boxed areas denote locations of dorsal (P) and ventral (Q) high-magnification images. T UMAP plot of Crym expression in the scRNA-Seq neurogenic lineage. (U-V) Confocal micrographs of Crym RNA (magenta), DCX (green), and GFAP (white) protein expression in the dorsal (U) and ventral (V) V-SVZ. (W-X). Quantification of DAPI+,S100B- Crym RNAscope puncta along the length of the V-SVZ (as in M: 0 = ventral-most extent, 1 = dorso-lateral-most extent of the wedge; n=3, bregma~ 1.42, 1.18 and 0.98 mm). X. Summary schematic of Crym expression in three rostro-caudal coronal sections (~bregma 1.50, 0.98 and 0.14 mm); strong expression (magenta); low expression (white). Boxed areas denote locations of dorsal (U) and ventral (V) high-magnification images. DAPI: blue, LV: lateral ventricle, d: dorsal, v: ventral, CC: corpus callosum, Str: striatum. Scale bars: 10 µm (K, L, P, Q, U, and V). Figure 3—source data 1.Quantifications of Crym, Dio2, and Urah RNAscope spots.

Fig 3: Whole mounts reveal CRYM expression in a wide ventral domain.(A-C) Confocal micrograph of a hGFAP:GFP coronal mouse brain section, where the V-SVZ is immunostained for GFP (green) and CRYM (magenta). B. High-magnification image of the dorsal wedge region of the V-SVZ. (C) High-magnification image of the ventral V-SVZ. (D-G) Immunostaining of CRYM (magenta) in a whole-mount preparation of the lateral wall of the V-SVZ, co-stained with ß-CATENIN (white), with a summary schematic depicting the extent of the CRYM+ domain. (E–G) Higher magnification images of boxed regions in (D) showing the distribution of CRYM+ (magenta) in V-SVZ cells outlined by ß-CATENIN (white). (H-I) High-magnification images of GFP+ (green) B1 cell-containing pinwheels in the dorsal (H) and ventral (I) V-SVZ outlined by ß-CATENIN (white), also immunostained for CRYM (magenta). Quantifications showed that 95.11% ± 2.65 (SD) of the GFP+ B1 cells were CRYM+ in the ventral domain of the V-SVZ. In contrast, only 4.71% ± 1.38 (SD) of the GFP+ B1 cells in the dorsal region were CRYM+ (n=3; T-test, p<0.0001). (J-K) Immunogold transmission electron micrographs of CRYM+ B1 cell in the V-SVZ. K. High magnification of B1 cell apical contact with the lateral ventricle. A: anterior, P: posterior, D: dorsal, V: ventral. Scale bars: 150 µm (A), 20 µm (B and C), 200 µm (D), 50 µm (E, F and G), 10 µm (H and I), 2 µm (J), and 500 nm (K). Figure 4—source data 1.Quantifications of CRYM+GFP+ B cells in coronal sections and whole mounts.

Fig 4: Contractile properties of Crym tg muscle. Force frequency curves (A, B, D, E, H) of gastrocnemius (A, B), soleus (D, E), and extensor digitorum longus (EDL) (H). Force was normalized to gastrocnemius muscle weight (A) or to soleus or EDL cross-sectional area (D, H). For fatigue, force was normalized to peak force (B, E). Ca2+ transients in isolated myofibers (C, F, G). C. Representative Ca2+ transient evoked by a voltage pulse, visualized with Rhod2. Blue, control; red, Crym tg. F. Maximal amplitudes of the Ca2+ transients. (n = 100). G. Mean time constants for the decay of the transients (n = 100). Statistics utilized the Student's t-test for Ca2+ measurements (F, G) or the Holm-Šídák test on the gastrocnemius (n = 5; A, B), soleus (control n = 4, Crym tg n = 5; D, E), or EDL (control n = 6, Crym tg n = 5; H) at a = 0.05. All error bars show standard error. The results show no statistically significant differences in any of these measurements.

Fig 5: Immunolabeling of µ-crystallin in control and Crym tg skeletal muscles. A-D, Cross (A–D), longitudinal sections (E–H), of Crym tg (A, C, E, G, H) and control (B, D, F) TA (A, B, E-H) and soleus muscles (C, D) were stained with anti-µ-crystallin antibody and Alexa Fluor-568-conjugated secondary antibody. In G-H, longitudinal sections of Crym tg TA muscle (G, µ-crystallin only) were colabeled with anti-desmin and Alexa Fluor-488-conjugated secondary antibody (H, yellow color shows colabeled structures). I, J, Flexor digitorum brevis (FDB) myofibers in culture from Crym tg (I) and control mice (J) were labeled with anti-µ-crystallin antibody and Alexa-Fluor 488-conjugated secondary antibody. The results show that µ-crystallin was detected at higher levels in tg muscles than in controls. In TA muscle and FDB myofibers it was enriched at the levels of the sarcolemma (arrows, A,E,G, I) and Z-disks, colabeled with desmin (arrowhead, H) or shown without desmin colabel (arrowhead, I). Inset panels (H–J) are brightened and magnified. Crym tg (I) and control (J) FDB images were brightened and magnified equivalently. A-F, scale bars = 20 µm; G, H, scale bars = 5 µm; I, J, scale bars = 10 µm.