Journal: Scientific Reports

Article Title: Identification of the optic recess region as a morphogenetic entity in the zebrafish forebrain

doi: 10.1038/srep08738

Figure Lengend Snippet: (A–C): Single confocal plane of a 48 hpf embryonic forebrain stained with DAPI (gray), immunolabeled for ZO-1 (A and B) or acetylated α-tubulin (C), in ventral (A and left panel in C) and lateral (B and right panel in C) views. The telencephalon (Tel), the optic recess region (ORR), and the hypothalamus (Hyp) form three distinct cellular regions in the secondary prosencephalon. The ORR is bordered by the dense fiber bundles, the anterior commissure (ac) and post-optic commissure (poc). The asterisk (*) in (B) (lateral view) indicates the enlargement of the dorsal ventricular lumen corresponding to the anterior intraencephalic sulcus (AIS). (D): Surface rendering of the shape of ventricle (white, in opacity), reconstructed from ZO-1 immunolabeling, overlapped on the shape of the brain (white, in transparency), reconstructed from DAPI staining. V = ventral, D = dorsal, L = lateral and F = frontal views. The 3D representation of both ventricle and brain facilitates the visualization of the convoluted ventricular organization in the forebrain. (E–F): Single confocal plane of a 48 hpf embryonic forebrain labeled for the neurogenic markers ccna2 , elavl3 and HuC/D in ventral (E) and lateral (F) views. Bottom left drawings show the level of corresponding optical planes. ccna2 -positive proliferative cells are concentrated around the ventricular zones, while elavl3 -positive differentiating and HuC/D-positive differentiated cells are located at the periphery of the neural tube. Scale bars = 50 μm.

Article Snippet: Mouse anti-ZO-1 (clone ZO1-1A12, Invitrogen) was used at a dilution of 1/200 to label the ventricles .

Techniques: Staining, Immunolabeling, Labeling

Journal: Scientific Reports

Article Title: Identification of the optic recess region as a morphogenetic entity in the zebrafish forebrain

doi: 10.1038/srep08738

Figure Lengend Snippet: (A–B): Single confocal plane of a 30 hpf embryonic forebrain stained with DAPI (gray) and immunolabeled for ZO-1, in ventral (A) and lateral (B) views. (C): Surface rendering of the shape of ventricle (white, in opacity), reconstructed from ZO-1 immunolabeling, overlapped on the shape of the brain (white, in transparency), reconstructed from DAPI staining. V = ventral, D = dorsal, L = lateral and F = frontal views. At 30 hpf the organization of the ventricular system is less complex than at 48 hpf. The frontal view displays a characteristic keyhole shape of the ventricle, with a larger lateral expansion of the ventral part compared to the dorsal part. (D–E): Single confocal plane of a 30 hpf embryonic forebrain labeled with the neurogenic markers ccna2 , elavl3 and HuC/D, in ventral (D) and lateral (E) views. Bottom left drawings show the level of the corresponding optical plane. The ccna2 staining is located around the ventricular walls but in wider territories than at 48 hpf. The elavl3 staining is detected adjacent to the ccna2 staining, also in wider territories than at 48 hpf. Accordingly, much fewer HuC/D-positive cells are detected than at 48 hpf, and they are all distributed at the periphery of the neural tube. The arrowheads in D indicate Hu-expressing cells located at the boundaries of telencephalon/ORR and ORR/hypothalamus. Scale bars = 50 μm.

Article Snippet: Mouse anti-ZO-1 (clone ZO1-1A12, Invitrogen) was used at a dilution of 1/200 to label the ventricles .

Techniques: Staining, Immunolabeling, Labeling, Expressing

Journal: Scientific Reports

Article Title: Identification of the optic recess region as a morphogenetic entity in the zebrafish forebrain

doi: 10.1038/srep08738

Figure Lengend Snippet: (A–B): Single confocal plane of a 24 hpf embryonic forebrain stained with DAPI (gray) and immunolabeled for ZO-1, in ventral (A) and lateral (B) views. diencephalon (Die); hypothalamus (Hyp); optic recess region (ORR); telencephalon (Tel). (C): Surface rendering of the shape of ventricle (white, in opacity), reconstructed from ZO-1 immunolabeling, overlapped on the shape of the brain (white, in transparency), reconstructed from DAPI staining. V = ventral, D = dorsal, L = lateral and F = frontal views. At 24 hpf the organization of the ventricular system is considerably simpler than at 48 hpf. The telencephalic ventricle rostral to the optic recess is not yet expanded (in contrast to 30 hpf). (D–E): Single confocal plane of a 24 hpf embryonic forebrain labeled for the neurogenic markers ccna2 , elavl3 and HuC/D, in ventral (D) and lateral (E) views. Bottom left drawings show the level of corresponding optical plane. The ccna2 staining is widely distributed while elavl3 and especially HuC/D expressions are restricted to small peripheral territories. The arrow heads in D indicate Hu-expressing cells located at the boundaries of telencephalon/ORR and ORR/hypothalamus. Scale bars = 50 μm.

Article Snippet: Mouse anti-ZO-1 (clone ZO1-1A12, Invitrogen) was used at a dilution of 1/200 to label the ventricles .

Techniques: Staining, Immunolabeling, Labeling, Expressing

Journal: Developmental biology

Article Title: Yap is essential for retinal progenitor cell cycle progression and RPE cell fate acquisition in the developing mouse eye

doi: 10.1016/j.ydbio.2016.09.001

Figure Lengend Snippet: RPE transdifferentiates into retina in the absence of Yap. IF analysis of the RPE and retina from WT (A, C, E, G, J and L) and Yap CKO (B, D, F, H, I, K and M) at E16.5. (A) Otx2-positive RPE cells (arrows) show a regularly spaced, single-layered arrangement while retinal photoreceptor cells show an apically-enriched, scattered pattern (arrowheads). RPE and RPE derived tissues are marked by dashed lines. Note that the single-layered Otx2 positive pattern in WT RPE changes into that of retina in Yap-deficient eyes. (C & D) Ezrin (green), a marker for apical villi of RPE, is altered or absent in duplicated multilayered epithelium. Zo1 (red) marks the tight junction of the retinal and RPE epithelia. (E & F) Retinal progenitor gene, Chx10, is ectopically expressed in the multilayered tissue induced by Yap-deficiency. (G, H & I) Expression of RPE fate regulating genes, Sox9 and Mitf, is altered or absent in ectopic, multilayered epithelium. (J & K) Duplicated multilayer tissue expresses ectopic neuronal marker, β-Tubulin III, which is normally absent in WT RPE (green). Pax6 (+) cells (red) are ectopically located at the basal side of the duplicated epithelia. (L & M) Aberrant Pax2 (+) cells (red), an optic stalk marker, are scattered in the ectopic epithelium in Yap CKO (arrows). Hoechst 33258 was used for nuclear counter staining (blue). Le; lens, Scale bars; 50 μm.

Article Snippet: Primary antibodies used to stain tissue sections were aPKC (#610207; Becton Dickinson), BrdU (#347580; BD and ab6326; Abcam), α-Catenin (610193; BD), Chx10 (X1179P; Exalpha Biologicals), CC3 (#9661; Cell Signaling Technology (CST)), Pan-Crb (synthesized using VGARVPPTPNLKLPPEERLI), Ezrin (ab4069; Abcam), GFP (GFP-1020; Aves), Lamin B1 (#9087; CST), Luciferase (ab21176; Abcam), Mitf (#x2398M; Exalpha), Otx2 (ab9566; Millipore), p27 (#610241; BD), Pals1 (07-708; Millipore), Pax2 (PRB-276P; Covance), Pax6 (RBP-278P; Covance), PCNA (#2586; CST), pH3 (#06-570; Millipore), rhodopsin (#1840-RHO; Phosphosolutions), Sox9 (AB5809; Millipore), β-Tubulin III (MMS-435P; Covance), Yap (#4912; CST and ab56701; Abcam), pYap (#4911; CST) and Zo1 (#610966; BD).

Techniques: Derivative Assay, Marker, Expressing, Staining

Journal: PLoS ONE

Article Title: The Structural and Functional Organization of the Podocyte Filtration Slits Is Regulated by Tjp1/ZO-1

doi: 10.1371/journal.pone.0106621

Figure Lengend Snippet: (A) Kidney sections from Tjp1 flox/flox and Nphs1-Cre: Tjp1 flox/flox mice were examined with the antibody against Tjp1. Tjp1 was specifically eliminated from podocytes, while it was detected in endothelial cells in the glomerulus in the Nphs1-Cre: Tjp1 flox/flox mice (arrow). The expression of Tjp1 in the Bowman's capsule epithelial cells (double arrows) and in the renal tubules (arrowheads) was also unaffected. See also . Scale bars, 10 µm. (B–I) Analyses of the control ( Tjp1 flox/flox ) and Tjp1 △pod ( Nphs1-Cre: Tjp1 flox/flox ) mice at 6 weeks of age. The Tjp1 △pod mice exhibited significant growth retardation (B) and developed massive proteinuria (C). (D) Gross appearance of the kidneys from the control and Tjp1 △pod mice at 6 weeks of age. (E) Histological analyses with periodic acid-Schiff (PAS) staining displayed disorder in the tissue architecture of the Tjp1 △pod mice kidney (top panels). Severe glomerulosclerosis and proteinaceous casts in the dilated renal tubules were observed in the Tjp1 △pod mice, but not in control mice (bottom panels). Scale bars, 200 µm (top panels), 20 µm (bottom panels). (F) The number of sclerosed glomeruli was expressed as a percentage of the total number of glomeruli (mean ± SEM of n = 3, **p <0.001). (G) Transmission electron micrographs showing the loss of slit diaphragms, destruction of foot processes, and aberrant glomerular basement membranes in the Tjp1 △pod mice. Scale bars, 2 µm (top panels), 0.5 µm (bottom panels). (H and I) Histopathological analyses by Jones' silver (H) and Masson's trichrome (I) stain. Global sclerosis with extensive deposits of basement membrane components were observed with both stains. Scale bars, 10 µm.

Article Snippet: The following primary antibodies were used: rat anti-Tjp1 (Santa Cruz Biotech), rabbit anti-Tjp1 (Invitrogen), rabbit anti-Tjp2 (Invitrogen), rabbit anti-Tjp3 (Invitrogen), rabbit anti-WT1 (Santa Cruz Biotech), goat anti-VE-cadherin (Santa Cruz Biotech), rabbit atni-Caludin2 (Invitrogen), guinea pig anti-Nephrin (Progen), rabbit anti-Podocin (Sigma-Aldrich), rabbit anti-Fyn (Sigma-Aldrich), rabbit anti-Synaptopodin (Sigma-Aldrich), rabbit anti-α-actinin-4 (Millipore), rabbit anti-CD2AP (Cell signaling), and rabbit anti-β-actin (Sigma-Aldrich).

Techniques: Expressing, Staining, Transmission Assay

Journal: PLoS ONE

Article Title: The Structural and Functional Organization of the Podocyte Filtration Slits Is Regulated by Tjp1/ZO-1

doi: 10.1371/journal.pone.0106621

Figure Lengend Snippet: (A–C) SEM analyses of glomeruli from the control and Tjp1 △pod mice. Foot processes were severely disorganized in the Tjp1 △pod mice at 6 weeks of age (A). The loss of interdigitation and the impaired adhesion to the GBM of the podocyte foot processes was detected in the Tjp1 △pod mice at 2 weeks of age (B). At an earlier time point, 1 week of age, the foot processes were attached to the GBM but the interdigitation was disorganized in the Tjp1 △pod mice (C). Scale bars, 2 µm (A, B, C top panels), 0.5 µm (C bottom panels). (D) Immunofluorescence analyses of podocyte components: nephrin, podocin, Fyn, and synaptopodin (Synpo). The distribution of the slit diaphragm membrane proteins, nephrin and podocin, was fragmented and the signal intensity was reduced in the Tjp1 △pod mice at 1 week and 2 weeks of age. The signal of the actin-binding protein Synpo was increased in the Tjp1 △pod mice at 2 weeks of age. Fyn, a member of src family kinase, was not affected. See also . Scale bars, 10 µm.

Article Snippet: The following primary antibodies were used: rat anti-Tjp1 (Santa Cruz Biotech), rabbit anti-Tjp1 (Invitrogen), rabbit anti-Tjp2 (Invitrogen), rabbit anti-Tjp3 (Invitrogen), rabbit anti-WT1 (Santa Cruz Biotech), goat anti-VE-cadherin (Santa Cruz Biotech), rabbit atni-Caludin2 (Invitrogen), guinea pig anti-Nephrin (Progen), rabbit anti-Podocin (Sigma-Aldrich), rabbit anti-Fyn (Sigma-Aldrich), rabbit anti-Synaptopodin (Sigma-Aldrich), rabbit anti-α-actinin-4 (Millipore), rabbit anti-CD2AP (Cell signaling), and rabbit anti-β-actin (Sigma-Aldrich).

Techniques: Immunofluorescence, Binding Assay

Journal: PLoS ONE

Article Title: The Structural and Functional Organization of the Podocyte Filtration Slits Is Regulated by Tjp1/ZO-1

doi: 10.1371/journal.pone.0106621

Figure Lengend Snippet: (A and B) The glomerular lysates from the control and Tjp1 △pod mice at 1 week and 2 weeks of age were analyzed by Western blotting with the antibodies against podocyte components (A). The results were quantified (B). Nephrin and podocin were significantly downregulated in the Tjp1 △pod mice both at 1 week and 2 weeks of age. The considerable increase in the Synpo protein was observed in 2-week-old Tjp1 △pod mice. The expression level of CD2AP, Fyn, and α-actinin4 (ACTN4) did not exhibit differences between the control and Tjp1 △pod mice. (C) mRNA expression of podocyte components was determined by qPCR. The difference in the neprhin and podocin mRNA levels between the control and Tjp1 △pod mice at 1 week and 2 weeks of age did not correlate with the reduction in the protein levels of those molecules. The mRNA level of most other molecules did not show these alterations, although some of them did exhibit upregulation without an increase in protein levels except for Synpo at 2 weeks of age. (D and E) The protein expression of the podocyte components in the developing glomerulus was examined by Western blotting. The kidney lysates from the control and Tjp1 △pod mice at P0 and P3 were probed with antibodies against podocyte components (D). The quantification data indicated that podocin was downregulated at P0 and P3, while the reduction of nephrin protein was detected at P3. All data are represented as mean ± SEM of n = 3. *p <0.01, **p <0.001

Article Snippet: The following primary antibodies were used: rat anti-Tjp1 (Santa Cruz Biotech), rabbit anti-Tjp1 (Invitrogen), rabbit anti-Tjp2 (Invitrogen), rabbit anti-Tjp3 (Invitrogen), rabbit anti-WT1 (Santa Cruz Biotech), goat anti-VE-cadherin (Santa Cruz Biotech), rabbit atni-Caludin2 (Invitrogen), guinea pig anti-Nephrin (Progen), rabbit anti-Podocin (Sigma-Aldrich), rabbit anti-Fyn (Sigma-Aldrich), rabbit anti-Synaptopodin (Sigma-Aldrich), rabbit anti-α-actinin-4 (Millipore), rabbit anti-CD2AP (Cell signaling), and rabbit anti-β-actin (Sigma-Aldrich).

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: The Structural and Functional Organization of the Podocyte Filtration Slits Is Regulated by Tjp1/ZO-1

doi: 10.1371/journal.pone.0106621

Figure Lengend Snippet: (A) Nephrin and podocin were introduced into L cells with or without Tjp1. The expression of the transfected genes was examined by Western blotting of the cell lysastes and the protein complex formation was analyzed by the immunoprecipitation assay (IP) using anti-Tjp1 antibody or control IgG. (B and C) Cells stably expressing nephrin and podocin with or without Tjp1 were treated with 10 µM cycloheximide for an indicated amount of time. The protein levels of nephrin and podocin were analyzed by Western blotting (B) and the results were quantified (C). The relative expression of the band intensity at time 0 for each protein was defined as 100%. Data shows mean ± SEM of n = 3. (D) Mouse podocytes were seeded on the type-I collagen-coated dishes or non-coated petri dishes and cultured for 16 h, followed by the examination of the expression of Synpo by Western blotting. (E and F) Kidney sections from the control and Tjp1 △pod mice at P0 were stained with anit- podocin antibody. (E) Before the maturing stage, podocin was detected around the nucleus (arrowheads), in addition, at the basolatral membrane domains as dots (arrows) both in the control and Tjp1 △pod mice. (F) The distribution of podocin exhibited a linear pattern at the maturing stage in control mice (arrows in control), suggesting the formation of slit diaphragms. On the other hand, podocin was not integrated into a linear pattern in the Tjp1 △pod mice at the maturing stage (arrows in Tjp1 △pod ). Scale bars, 10 µm in the top panels and 2 µm in the bottom panels. (G) Transmission electron micrographs of P0 mouse podocytes demonstrating the presence of slit diaphragms at the maturing stage in the control mice (arrows in the top panel), but not in the Tjp1 △pod mice (arrows in the bottom panel). Scale bars, 0.2 µm. (H) Albumin and higher molecular weight proteins were detected in urine of P0 Tjp1 △pod mice.

Article Snippet: The following primary antibodies were used: rat anti-Tjp1 (Santa Cruz Biotech), rabbit anti-Tjp1 (Invitrogen), rabbit anti-Tjp2 (Invitrogen), rabbit anti-Tjp3 (Invitrogen), rabbit anti-WT1 (Santa Cruz Biotech), goat anti-VE-cadherin (Santa Cruz Biotech), rabbit atni-Caludin2 (Invitrogen), guinea pig anti-Nephrin (Progen), rabbit anti-Podocin (Sigma-Aldrich), rabbit anti-Fyn (Sigma-Aldrich), rabbit anti-Synaptopodin (Sigma-Aldrich), rabbit anti-α-actinin-4 (Millipore), rabbit anti-CD2AP (Cell signaling), and rabbit anti-β-actin (Sigma-Aldrich).

Techniques: Expressing, Transfection, Western Blot, Immunoprecipitation, Stable Transfection, Cell Culture, Staining, Transmission Assay, Molecular Weight