Fig 2: Laser confocal fluorescence microscopic images.The impact of DPSC-CM on trigeminal ganglion survival and neurite length after 4 days of culture in the presence of specific neutralising antibodies. Antibodies against GDNF (1 µg/mL; R&D systems, Oxon, UK; AF-212-NA) and NT-3 (2 µg/mL; R&D systems; Cat# AF1404) were used to test the impact of their absence on TG neuronal survival using NeuN (neuronal nucleus immune stain marker; Cat# ab128886 at a 1:1000 dilution; Abcam, Cambridge, MA, USA) and neurite outgrowth extension using MAP-2 (mature neuronal immune stain marker; Cat# ab11267 at a 1:500 dilution; Abcam). Immunolocalization of neuronal markers NeuN in neuronal cell body and MAP-2 in neuronal cell body and processes showing the difference in the number of surviving neurons and the neurite length, respectively. DAPI (blue) was used as a nuclear staining. Anti-GDNF antibody completely blocks CM mediated neurite outgrowth effect while anti-NT-3 markedly reduce the number of surviving neurons. Scale bars: 100 µm. Unpublished data. CM: Concitioned medium; DAPI: 4',6-diamidino-2-phenylindole; GDNF: glial cell line-derived neurotrophic factor; MAP-2: microtubule-associated protein 2; NeuN: neuronal nuclear protein; NT-3: neurotrophin-3.

Fig 3: Akt and GSK3ß phosphorylation in Pten-deficient mice.(A) Phosphorylation of Akt and GSK3ß was measured by immunofluorescence staining using anti-phospho-Akt (Ser473) (pAkt) and anti-phospho-GSK3ß (Ser9) (pGSK3ß) antibodies at E13.5. Compared to wild-type spiral ganglia, Akt phosphorylation (green) increased substantially in Pten-deficient mice (a–f). GSK3ß phosphorylation (green) was also highly expressed in the spiral ganglia of Pten-deficient mice compared to that in wild-type mice (g–l). In the absence of Pten, all pAkt and pGSK3ß cells were maintained in Islet-1 positive cells (red) (a–l). Wild-type mice showed well-organized patterns of Islet-1-positive cells (a–c), whereas Pten-deficient mice showed a slightly scattered form of spiral ganglia (d–f). Cleaved caspase-3-positive apoptotic neurons (red) were sometimes co-localized with TrkC-positive cells (green) (arrowheads in r). DAPI-stained nuclei (blue) are seen in all images. Scale bar: 100 µm. (B) Western blotting analysis of Pten, pAkt, Akt, pGSK3ß, GSK3ß, and ß-actin expression in the inner ear at E14.5. Proteins were extracted from four pooled inner ears at E14.5. (C) The relative intensity of each phospho-protein was normalized to the total level of the same protein. Levels of both pAkt (a) and pGSK3ß (b) were significantly increased in Pten cKO mice (4 cochleae, P<0.05).

Fig 4: Neuronal apoptosis in the cochleovestibular ganglion (CVG) complex of Pten-deficient mice.(A–D) Apoptotic cells in the CVG were stained with anti-cleaved caspase-3 antibody at E11.5. Cleaved caspase-3 immunoreactivity (red) increased substantially in Pten-deficient mice compared to that in wild-type mice. (A) Apoptotic neurons were occasionally co-localized with TrkC-positive (green) (arrows in f) or negative cells. Higher magnification images of d, é, and f are shown in the insets in d, e, and f, respectively. Scale bars: 100 µm. (B) Proliferating PCNA-positive cells (green) were not seen in apoptotic neurons (arrows in f ´). Higher magnification images of d, e, and f are shown in the insets in d, e, and f, respectively. Scale bars: 100 µm. (C) Cleaved caspase-3-positive cells (red) were also Islet1-negative cells (arrows in f ´). Apoptotic neurons (red) were distributed in the core of the non-proliferative area that expressed Islet1 (green). Higher magnification images of d, e, and f are shown in the insets in d, e, and f, respectively. Scale bars: 100 µm. (D) Numbers of cleaved caspase-3-positive apoptotic cells in the CVG were significantly increased in Pten-deficient mice at E11.5 (3 cochleae, P<0.001).

Fig 5: Ultrastructural Analysis of Ntrk3 Mutant Kidneys.Kidneys from Ntrk3-/- (') and control littermates at 4 days of age were examined by scanning (A, B) and transmission electron microscopy (C, D). A, B) Note the disorganized patterning and irregularly shaped primary and secondary foot processes. C, D) Note the fusion of foot processes and the lack of well-spaced slit diaphragms in Ntrk3 mutants in D. Scale bars are 1 µm in A and B, 2 µm in C, and 500 nm in D.