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goat anti netrin 1 (R&D Systems)

Name: goat anti netrin 1
Brand: R&D Systems
Rating: 94 (50 reviews)

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R&D Systems goat anti netrin 1
Goat Anti Netrin 1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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( A ) OVCAR8 cells were allowed to form spheroids for 6 hr before fixation in formalin and embedding in paraffin for sectioning. Serial sections were stained with Netrin-1 Abs or a no primary antibody control. ( B ) OVCAR8 cells overexpressing Netrin-1 were used to prepare spheroids as in A and stained in parallel to samples in A. ( C ) OVCAR8 deleted for Netrin-1 expression were used to prepare spheroids as in A and serial sections were stained for Netrin-1 or with a no primary antibody control and counter stained. ( D ) OVCAR8 cells were allowed to form spheroids for 6 hr before fixation in formalin and embedding in paraffin for sectioning. Serial sections were stained with Netrin-3 Abs or a no primary antibody control. ( E ) OVCAR8 cells overexpressing Netrin-3 were used to prepare spheroids as in D and stained in parallel to samples in D. ( F ) OVCAR8 deleted for Netrin-3 expression were used to prepare spheroids as in D and serial sections were stained for Netrin-3 or with a no primary antibody control. All scale bars = 100 μm.

Journal: eLife

Article Title: Netrin signaling mediates survival of dormant epithelial ovarian cancer cells

doi: 10.7554/eLife.91766

Figure Lengend Snippet: ( A ) OVCAR8 cells were allowed to form spheroids for 6 hr before fixation in formalin and embedding in paraffin for sectioning. Serial sections were stained with Netrin-1 Abs or a no primary antibody control. ( B ) OVCAR8 cells overexpressing Netrin-1 were used to prepare spheroids as in A and stained in parallel to samples in A. ( C ) OVCAR8 deleted for Netrin-1 expression were used to prepare spheroids as in A and serial sections were stained for Netrin-1 or with a no primary antibody control and counter stained. ( D ) OVCAR8 cells were allowed to form spheroids for 6 hr before fixation in formalin and embedding in paraffin for sectioning. Serial sections were stained with Netrin-3 Abs or a no primary antibody control. ( E ) OVCAR8 cells overexpressing Netrin-3 were used to prepare spheroids as in D and stained in parallel to samples in D. ( F ) OVCAR8 deleted for Netrin-3 expression were used to prepare spheroids as in D and serial sections were stained for Netrin-3 or with a no primary antibody control. All scale bars = 100 μm.

Article Snippet: Antibodies used for blotting were p42/44 MAPK (Erk1/2) (Cell Signaling Technology rabbit antibody #4695), Phospho-p44/42 MAPK(Erk1/2) T202/Y204 (Cell Signaling Technology; rabbit antibody #4370), p38 MAPK (Cell Signaling Technology; rabbit antibody #9212), p38 MAPK T180/Y182 (Cell Signaling Technology; rabbit antibody #9211), p130(RBL2, Santa Cruz Biotechnology, rabbit antibody sc-317 discontinued), Ki67 (Abcam rabbit antibody, # ab16667), DYRK1A (Cell Signaling Technology anti-DYRK1A rabbit antibody #8765), Netrin-1 (Abcam rabbit antibody #ab126729), Netrin-3 (Abcam rabbit antibody #ab185200), α-tubulin (Cell Signaling Technology anti-αTubulin rabbit antibody #2125) and β-actin (Millipore Sigma rabbit antibody #A2066).

Techniques: Staining, Control, Expressing

( A ) Illustration of netrin ligands, receptors, and other intracellular signaling molecules that are included in the Axon Guidance pathway category. The frequency of their identification in CRISPR screens is illustrated by shading and indicates how many cell lines had an enrichment ratio <1 for a given component. ( B ) The indicated ovarian cancer cell lines were infected with lentiviruses expressing sgRNAs directed against the indicated Netrin signaling genes. Cells were transferred to suspension culture conditions to induce spheroid formation for 72 hr and then returned to adherent conditions for 24 hr to facilitate reattachment. Re-attached cells were stained with Crystal Violet and retained dye was extracted and quantitated to measure relative survival. Each cell-gene combination was assayed in at least three biological replicates, averaged, and viability is displayed as a bubble plot. Mean survival for a given cell-gene combination was compared with GFP control gRNA transduced cells using one way anova and significance levels are illustrated by bubble size. Inviable cell-gene combinations are depicted as empty spaces. ( C ) Cultures of the indicated cell lines were cultured in suspension for five days and stimulated with 0.5 μg/mL Netrin-1. Netrin-1 signaling was analyzed by SDS-PAGE and western blotting for phospho-ERK, ERK, and tubulin. ( D ) Quiescent adherent OVCAR8 cells were stimulated with 0.5 μg/mL Netrin-1 or 0.5 μg/mL EGF, and compared with OVCAR8 cells in suspension stimulated as in C. Extracts were prepared and blotted for phospho-ERK, ERK, and tubulin. ( E ) Suspension cultures of OVCAR8, or knock out derivatives, were harvested and analyzed for relative phosphorylation levels of ERK by western blotting. Total ERK and tubulin blotting serve as expression and loading controls. ( F ) Netrin-1 signaling in OVCAR8, UNC5 4KO and DDN 3KO derivatives was tested by transferring cells to suspension and stimulating with Netrin-1 as before. Western blotting for phospho-ERK, ERK were also as before. ( G ) OVCAR8 cells were seeded in suspension culture and treated with the MEK inhibitors PD184352, Trametinib or DMSO vehicle for 72 hr. Mean viability was determined by re-attachment and compared by one way anova (****p<0.0001). ( H ) OVCAR8 cells were cultured in suspension and treated with Trametinib or DMSO vehicle as in G. Viability was determined by trypan blue dye exclusion and compared by one way anova (**p<0.01). ( I ) Extracts were prepared from Trametinib and control treated spheroid cells and blotted for phospho-ERK, ERK, and tubulin. ( J ) Model summarizing the roles of Netrin ligands, receptors, and downstream targets MEK and ERK in dormant survival signaling. Figure 5—source data 1. Numerical data used for bubble plot in . Figure 5—source data 2. Original files for western blot analysis in (pERK T202/Y204, ERK, Tubulin). Figure 5—source data 3. PDFs containing annotation of original western blots in (pERK T202/Y204, ERK, Tubulin). Figure 5—source data 4. Original files for western blot analysis in (pERK T202/Y204, ERK, Tubulin). Figure 5—source data 5. PDFs containing annotation of original western blots in (pERK T202/Y204, ERK, Tubulin). Figure 5—source data 6. Original files for western blot analysis in (pERK T202/Y204, ERK, Tubulin). Figure 5—source data 7. PDFs containing annotation of original western blots in (pERK T202/Y204, ERK, Tubulin). Figure 5—source data 8. Original files for western blot analysis in (pERK T202/Y204, ERK). Figure 5—source data 9. PDFs containing annotation of original western blots in (pERK T202/Y204, ERK). Figure 5—source data 10. Numerical data used for graphs in . Figure 5—source data 11. Original files for western blot analysis in (pERK T202/Y204, ERK, Tubulin). Figure 5—source data 12. PDFs containing annotation of original western blots in (pERK T202/Y204, ERK, Tubulin).

Journal: eLife

Article Title: Netrin signaling mediates survival of dormant epithelial ovarian cancer cells

doi: 10.7554/eLife.91766

Figure Lengend Snippet: ( A ) Illustration of netrin ligands, receptors, and other intracellular signaling molecules that are included in the Axon Guidance pathway category. The frequency of their identification in CRISPR screens is illustrated by shading and indicates how many cell lines had an enrichment ratio <1 for a given component. ( B ) The indicated ovarian cancer cell lines were infected with lentiviruses expressing sgRNAs directed against the indicated Netrin signaling genes. Cells were transferred to suspension culture conditions to induce spheroid formation for 72 hr and then returned to adherent conditions for 24 hr to facilitate reattachment. Re-attached cells were stained with Crystal Violet and retained dye was extracted and quantitated to measure relative survival. Each cell-gene combination was assayed in at least three biological replicates, averaged, and viability is displayed as a bubble plot. Mean survival for a given cell-gene combination was compared with GFP control gRNA transduced cells using one way anova and significance levels are illustrated by bubble size. Inviable cell-gene combinations are depicted as empty spaces. ( C ) Cultures of the indicated cell lines were cultured in suspension for five days and stimulated with 0.5 μg/mL Netrin-1. Netrin-1 signaling was analyzed by SDS-PAGE and western blotting for phospho-ERK, ERK, and tubulin. ( D ) Quiescent adherent OVCAR8 cells were stimulated with 0.5 μg/mL Netrin-1 or 0.5 μg/mL EGF, and compared with OVCAR8 cells in suspension stimulated as in C. Extracts were prepared and blotted for phospho-ERK, ERK, and tubulin. ( E ) Suspension cultures of OVCAR8, or knock out derivatives, were harvested and analyzed for relative phosphorylation levels of ERK by western blotting. Total ERK and tubulin blotting serve as expression and loading controls. ( F ) Netrin-1 signaling in OVCAR8, UNC5 4KO and DDN 3KO derivatives was tested by transferring cells to suspension and stimulating with Netrin-1 as before. Western blotting for phospho-ERK, ERK were also as before. ( G ) OVCAR8 cells were seeded in suspension culture and treated with the MEK inhibitors PD184352, Trametinib or DMSO vehicle for 72 hr. Mean viability was determined by re-attachment and compared by one way anova (****p<0.0001). ( H ) OVCAR8 cells were cultured in suspension and treated with Trametinib or DMSO vehicle as in G. Viability was determined by trypan blue dye exclusion and compared by one way anova (**p<0.01). ( I ) Extracts were prepared from Trametinib and control treated spheroid cells and blotted for phospho-ERK, ERK, and tubulin. ( J ) Model summarizing the roles of Netrin ligands, receptors, and downstream targets MEK and ERK in dormant survival signaling. Figure 5—source data 1. Numerical data used for bubble plot in . Figure 5—source data 2. Original files for western blot analysis in (pERK T202/Y204, ERK, Tubulin). Figure 5—source data 3. PDFs containing annotation of original western blots in (pERK T202/Y204, ERK, Tubulin). Figure 5—source data 4. Original files for western blot analysis in (pERK T202/Y204, ERK, Tubulin). Figure 5—source data 5. PDFs containing annotation of original western blots in (pERK T202/Y204, ERK, Tubulin). Figure 5—source data 6. Original files for western blot analysis in (pERK T202/Y204, ERK, Tubulin). Figure 5—source data 7. PDFs containing annotation of original western blots in (pERK T202/Y204, ERK, Tubulin). Figure 5—source data 8. Original files for western blot analysis in (pERK T202/Y204, ERK). Figure 5—source data 9. PDFs containing annotation of original western blots in (pERK T202/Y204, ERK). Figure 5—source data 10. Numerical data used for graphs in . Figure 5—source data 11. Original files for western blot analysis in (pERK T202/Y204, ERK, Tubulin). Figure 5—source data 12. PDFs containing annotation of original western blots in (pERK T202/Y204, ERK, Tubulin).

Techniques: CRISPR, Infection, Expressing, Suspension, Staining, Control, Cell Culture, SDS Page, Western Blot, Knock-Out, Transferring

( A ) RT-qPCR was performed on RNA isolated from control sgRNA expressing OVCAR8 cells and UNC5 4KO to assess expression of UNC5 family members. Graphs depict relative expression levels for each indicted gene in control and knock out cell lines. Mean expression levels of targeted cells were compared to background measurements using one way anova (****p<0.0001). ( B ) RT-qPCR was used to measure mRNA levels of NTN1 and NTN3 transcripts in their respective CRISPR targeted, and OVCAR8 controls. Mean values were compared by one way anova (****p<0.0001). ( C ) Levels of mRNA corresponding to DCC, DSCAM, and NEO1 were determined by RT-qPCR in DDN 3KO cells and control OVCAR8. Mean values were compared by one way anova (****p<0.0001). ( D ) The indicated genotypes of cells were subjected to suspension culture and protein extracts were prepared at the indicated times. Proteins were resolved by SDS-PAGE, blotted, and probed with the indicated antibodies. Figure 5—figure supplement 1—source data 1. Numerical data used for graphs in . Figure 5—figure supplement 1—source data 2. Original files for western blot analysis in (pDAPK1 S318, DAPK1, Tubulin). Figure 5—figure supplement 1—source data 3. PDF containing annotation of original western blots in (pDAPK1 S318, DAPK1, Tubulin).

Journal: eLife

Article Title: Netrin signaling mediates survival of dormant epithelial ovarian cancer cells

doi: 10.7554/eLife.91766

Figure Lengend Snippet: ( A ) RT-qPCR was performed on RNA isolated from control sgRNA expressing OVCAR8 cells and UNC5 4KO to assess expression of UNC5 family members. Graphs depict relative expression levels for each indicted gene in control and knock out cell lines. Mean expression levels of targeted cells were compared to background measurements using one way anova (****p<0.0001). ( B ) RT-qPCR was used to measure mRNA levels of NTN1 and NTN3 transcripts in their respective CRISPR targeted, and OVCAR8 controls. Mean values were compared by one way anova (****p<0.0001). ( C ) Levels of mRNA corresponding to DCC, DSCAM, and NEO1 were determined by RT-qPCR in DDN 3KO cells and control OVCAR8. Mean values were compared by one way anova (****p<0.0001). ( D ) The indicated genotypes of cells were subjected to suspension culture and protein extracts were prepared at the indicated times. Proteins were resolved by SDS-PAGE, blotted, and probed with the indicated antibodies. Figure 5—figure supplement 1—source data 1. Numerical data used for graphs in . Figure 5—figure supplement 1—source data 2. Original files for western blot analysis in (pDAPK1 S318, DAPK1, Tubulin). Figure 5—figure supplement 1—source data 3. PDF containing annotation of original western blots in (pDAPK1 S318, DAPK1, Tubulin).

Techniques: Quantitative RT-PCR, Isolation, Control, Expressing, Knock-Out, CRISPR, Suspension, SDS Page, Western Blot

( A ) TCGA RNA-seq data for HGSOC patients (TCGA PanCancer Atlas study) was used to identify high Netrin-1 or –3 and low Netrin-1 or –3 expressing patients (high expressing are above z-score 1.2). Overall survival was used to construct Kaplan-Meier plots and survival was compared using a logrank test. ( B ) OVCAR8 cells were stably transduced with lentiviral constructs to overexpress epitope tagged Netrin-1 or –3. Western blotting for Netrins, Myc-tags, and Actin were used to determine relative expression levels of both Netrins in these cell populations and vector controls. ( C ) Control and Netrin overexpressing cells were transferred to suspension culture conditions to form spheroids, and replated to assay for viability. Mean viability and standard deviation is shown for each. One-way anova was used to compare survival (* p<0.05, *** p<0.001). ( D ) Reattached spheroids were fixed and stained with Crystal Violet to examine size and abundance in control and Netrin-1 or –3 overexpression. Figure 7—source data 1. Original files for western blot analysis in (Myc, Netrin-1, Netrin-3, Actin). Figure 7—source data 2. PDFs containing annotation of original western blot analysis in (Myc, Netrin-1, Netrin-3, Actin). Figure 7—source data 3. Numerical data used for graphs in .

Journal: eLife

Article Title: Netrin signaling mediates survival of dormant epithelial ovarian cancer cells

doi: 10.7554/eLife.91766

Figure Lengend Snippet: ( A ) TCGA RNA-seq data for HGSOC patients (TCGA PanCancer Atlas study) was used to identify high Netrin-1 or –3 and low Netrin-1 or –3 expressing patients (high expressing are above z-score 1.2). Overall survival was used to construct Kaplan-Meier plots and survival was compared using a logrank test. ( B ) OVCAR8 cells were stably transduced with lentiviral constructs to overexpress epitope tagged Netrin-1 or –3. Western blotting for Netrins, Myc-tags, and Actin were used to determine relative expression levels of both Netrins in these cell populations and vector controls. ( C ) Control and Netrin overexpressing cells were transferred to suspension culture conditions to form spheroids, and replated to assay for viability. Mean viability and standard deviation is shown for each. One-way anova was used to compare survival (* p<0.05, *** p<0.001). ( D ) Reattached spheroids were fixed and stained with Crystal Violet to examine size and abundance in control and Netrin-1 or –3 overexpression. Figure 7—source data 1. Original files for western blot analysis in (Myc, Netrin-1, Netrin-3, Actin). Figure 7—source data 2. PDFs containing annotation of original western blot analysis in (Myc, Netrin-1, Netrin-3, Actin). Figure 7—source data 3. Numerical data used for graphs in .

Techniques: RNA Sequencing Assay, Expressing, Construct, Stable Transfection, Transduction, Western Blot, Plasmid Preparation, Control, Suspension, Standard Deviation, Staining, Over Expression

( A ) Oncoprint of genetic alterations in 10 netrin signaling related genes in 398 patients. Alteration types are defined on the right. Only the first 154 patients are shown. ( B ) Oncoprint illustrating gene expression outliers among 10 netrin signaling related genes in 201 patients (first 122 patients shown). Genomic data used is from Ovarian Serous Cystadenocarcinoma (TCGA, PanCancer Atlas) (201 samples with RNA seqV2, z-score cut off of 1.5). ( C ) TCGA RNA-seq data for HGSOC patients (TCGA PanCancer Atlas study) was used to identify high Netrin-1 or –3 and low Netrin-1 or –3 expressing patients (high expressing are above z-score 1.2). Progression free survival was used to construct Kaplan-Meier plots and survival was compared using a logrank test.

Journal: eLife

Article Title: Netrin signaling mediates survival of dormant epithelial ovarian cancer cells

doi: 10.7554/eLife.91766

Figure Lengend Snippet: ( A ) Oncoprint of genetic alterations in 10 netrin signaling related genes in 398 patients. Alteration types are defined on the right. Only the first 154 patients are shown. ( B ) Oncoprint illustrating gene expression outliers among 10 netrin signaling related genes in 201 patients (first 122 patients shown). Genomic data used is from Ovarian Serous Cystadenocarcinoma (TCGA, PanCancer Atlas) (201 samples with RNA seqV2, z-score cut off of 1.5). ( C ) TCGA RNA-seq data for HGSOC patients (TCGA PanCancer Atlas study) was used to identify high Netrin-1 or –3 and low Netrin-1 or –3 expressing patients (high expressing are above z-score 1.2). Progression free survival was used to construct Kaplan-Meier plots and survival was compared using a logrank test.

Techniques: Expressing, RNA Sequencing Assay, Construct

Journal: eLife

Article Title: Netrin signaling mediates survival of dormant epithelial ovarian cancer cells

doi: 10.7554/eLife.91766

Figure Lengend Snippet:

Techniques: Western Blot, Plasmid Preparation, Recombinant, CRISPR, Expressing, Selection, Sequencing, Gel Extraction, DNA Extraction, SYBR Green Assay, Protease Inhibitor, Software, Immunohistochemistry, Staining, Knock-Out, Kinase Assay, Construct, Over Expression, Amplification, Clone Assay, Control

A Western blot shows the decreased expression of Netrin-1 in the cortex of CKO mice. By Shapiro–Wilk test, the samples followed a normal distribution. Student’s t test, p < 0.05, * N = 3 for each. B Schematic representation of a sagittal section indicating the trajectory of the CST and the level of the coronal sections presented in this study. C – J Staining with anti-PKCγ antibody shows that the CST axons spread into two bundles, the medial and lateral one (arrowhead), in the ventral hindbrain of CKO mice ( C , D ). Besides, pyramidal decussation is much decreased ( E , F ), and PKCγ-stained area in the dorsal funiculus (arrow) is also reduced in CKO mice compared with that in control mice ( G – J ), J is enlargement of the boxed area in H . Arrowhead in H point to ectopic CST axons in the lateral funiculus of the CKO mice. I is quantification of PKCγ-stained area in the dorsal funiculus. By Shapiro–Wilk test, the samples followed a normal distribution. Student’s t test, p < 0.05, * N = 4 for each. K – T BDA is unilaterally injected into the right motor cortex and the CST is visualized by both PKCγ immunostaining (green) and BDA (red), showing the split pyramidal tract in the ventral hindbrain (arrowhead in L, N), reduced pyramidal decussation, and reduction of crossed CST axons in the dorsal funiculus (arrow), and uncrossed CST axons in the lateral funiculus (arrowhead in P , T ) of CKO mice. This experiment was replicated for three times. Boxed areas in O , P are enlarged in Q , S , and R , T , respectively. Scale bars = 100 μm in J , and 200 μm for the other panels. Cb cerebellum, cc corpus callosum, CST corticospinal tract, Ctx cerebral cortex, df dorsal funiculus, if lateral funiculus, py pyramidal tract.

Journal: Cell Death & Disease

Article Title: Ventricular Netrin-1 deficiency leads to defective pyramidal decussation and mirror movement in mice

doi: 10.1038/s41419-024-06719-1

Figure Lengend Snippet: A Western blot shows the decreased expression of Netrin-1 in the cortex of CKO mice. By Shapiro–Wilk test, the samples followed a normal distribution. Student’s t test, p < 0.05, * N = 3 for each. B Schematic representation of a sagittal section indicating the trajectory of the CST and the level of the coronal sections presented in this study. C – J Staining with anti-PKCγ antibody shows that the CST axons spread into two bundles, the medial and lateral one (arrowhead), in the ventral hindbrain of CKO mice ( C , D ). Besides, pyramidal decussation is much decreased ( E , F ), and PKCγ-stained area in the dorsal funiculus (arrow) is also reduced in CKO mice compared with that in control mice ( G – J ), J is enlargement of the boxed area in H . Arrowhead in H point to ectopic CST axons in the lateral funiculus of the CKO mice. I is quantification of PKCγ-stained area in the dorsal funiculus. By Shapiro–Wilk test, the samples followed a normal distribution. Student’s t test, p < 0.05, * N = 4 for each. K – T BDA is unilaterally injected into the right motor cortex and the CST is visualized by both PKCγ immunostaining (green) and BDA (red), showing the split pyramidal tract in the ventral hindbrain (arrowhead in L, N), reduced pyramidal decussation, and reduction of crossed CST axons in the dorsal funiculus (arrow), and uncrossed CST axons in the lateral funiculus (arrowhead in P , T ) of CKO mice. This experiment was replicated for three times. Boxed areas in O , P are enlarged in Q , S , and R , T , respectively. Scale bars = 100 μm in J , and 200 μm for the other panels. Cb cerebellum, cc corpus callosum, CST corticospinal tract, Ctx cerebral cortex, df dorsal funiculus, if lateral funiculus, py pyramidal tract.

Article Snippet: Membranes were then incubated overnight at 4 °C with the following primary antibodies: rabbit anti-Netrin-1 (1:1000, ab126729, Abcam) and rabbit anti-GAPDH (1:1000, LF206, Epizyme), followed by incubation with HRP-conjugated goat anti-rabbit (1:1000; KangCheng, China) for 2 hours at room temperature.

Techniques: Western Blot, Expressing, Staining, Injection, Immunostaining

A Netrin-1 mRNA is present in the VZ and dorsal midline region of hindbrain at E17.5 ( A ). B -B” Netrin-1 protein is not only present in the regions containing Netrin-1 mRNA, but also in the ventral hindbrain (arrow), where contains no mRNA at E17.5 (arrow in A ). Double immunostaining of Netrin-1 (red) and Glast (green) shows the localization of Netrin-1 in Glast-positive process of radial glia cells in the ventral medulla. B -B’ are the same filed, respectively. Boxed area in (B’) is enlarged in (B”). C -C” Double immunostaining of Netrin-1 (red) and 2H3 (green) shows overlapping of 2H3-positve CST axons and Netrin-1 protein in the ventral medulla at P0. Boxed area in (C’) is enlarged in (C”). D -D” The GFP signal is widely detected in the VZ of the hindbrain at E13.5 but excluded by the floor plate labeled by Shh . E , F The Netrin-1 expression in the VZ is overlapped with GFP signal at E13.5 ( E ) and P0 ( F ). Boxed area in E and F is enlarged in (E’) and (F’), respectively. The representative images were taken in the middle level of medulla oblongata where pyramidal tract accumulated in the ventral margin. Scale bars = 50 μm (E’, F’) and = 100 μm in other panels. 4 V 4th ventricle, VZ ventricular zone, fp floor plate, ml midline.

Journal: Cell Death & Disease

Article Title: Ventricular Netrin-1 deficiency leads to defective pyramidal decussation and mirror movement in mice

doi: 10.1038/s41419-024-06719-1

Figure Lengend Snippet: A Netrin-1 mRNA is present in the VZ and dorsal midline region of hindbrain at E17.5 ( A ). B -B” Netrin-1 protein is not only present in the regions containing Netrin-1 mRNA, but also in the ventral hindbrain (arrow), where contains no mRNA at E17.5 (arrow in A ). Double immunostaining of Netrin-1 (red) and Glast (green) shows the localization of Netrin-1 in Glast-positive process of radial glia cells in the ventral medulla. B -B’ are the same filed, respectively. Boxed area in (B’) is enlarged in (B”). C -C” Double immunostaining of Netrin-1 (red) and 2H3 (green) shows overlapping of 2H3-positve CST axons and Netrin-1 protein in the ventral medulla at P0. Boxed area in (C’) is enlarged in (C”). D -D” The GFP signal is widely detected in the VZ of the hindbrain at E13.5 but excluded by the floor plate labeled by Shh . E , F The Netrin-1 expression in the VZ is overlapped with GFP signal at E13.5 ( E ) and P0 ( F ). Boxed area in E and F is enlarged in (E’) and (F’), respectively. The representative images were taken in the middle level of medulla oblongata where pyramidal tract accumulated in the ventral margin. Scale bars = 50 μm (E’, F’) and = 100 μm in other panels. 4 V 4th ventricle, VZ ventricular zone, fp floor plate, ml midline.

Techniques: Double Immunostaining, Labeling, Expressing

A – D In situ hybridization shows that Netrin-1 transcripts are dramatically decreased in the VZ of Ntn1 Gfap CKO mice at E17.5 ( A , B ) and P0 ( C , D ) compared with controls. E – L Double staining of Netrin-1 (red) and 2H3 (green) shows that Netrin-1 protein in the ventral margin (asterisks) is located in the proximity of 2H3-labeled CST axons (triangles) of control mice, but it is obviously decreased in CKO mice at E17.5 ( E – H ) and P0 ( I – L ). E – H are the same fields, respectively. Scale bars = 100 μm. 4 V, 4th ventricle. I – L are the same fields, respectively. The inserts in G , H , K , L are enlarged boxed areas in (G’, H’, K’, L’). The representative images were taken at the caudal level of medulla oblongata where pyramidal tract accumulated in the ventral margin. This experiment was replicated in three different control and CKO mice. Scale bars = 100 μm. 4 V 4th ventricle, ml midline, VZ ventricular zone.

Journal: Cell Death & Disease

Article Title: Ventricular Netrin-1 deficiency leads to defective pyramidal decussation and mirror movement in mice

doi: 10.1038/s41419-024-06719-1

Figure Lengend Snippet: A – D In situ hybridization shows that Netrin-1 transcripts are dramatically decreased in the VZ of Ntn1 Gfap CKO mice at E17.5 ( A , B ) and P0 ( C , D ) compared with controls. E – L Double staining of Netrin-1 (red) and 2H3 (green) shows that Netrin-1 protein in the ventral margin (asterisks) is located in the proximity of 2H3-labeled CST axons (triangles) of control mice, but it is obviously decreased in CKO mice at E17.5 ( E – H ) and P0 ( I – L ). E – H are the same fields, respectively. Scale bars = 100 μm. 4 V, 4th ventricle. I – L are the same fields, respectively. The inserts in G , H , K , L are enlarged boxed areas in (G’, H’, K’, L’). The representative images were taken at the caudal level of medulla oblongata where pyramidal tract accumulated in the ventral margin. This experiment was replicated in three different control and CKO mice. Scale bars = 100 μm. 4 V 4th ventricle, ml midline, VZ ventricular zone.

Techniques: In Situ Hybridization, Double Staining, Labeling

A The body weight is comparable between control and CKO mice. By Shapiro–Wilk test, the samples followed a normal distribution. Student’s t test, p > 0.05, * N = 17 for control and N = 14 CKO mice. B , D No significant differences are found between the two groups in the traveled distance in the open-field ( B ), the time on the rods in the rotarod test ( C ) and the total time for mice landed to the floor in the pole test ( D ). E Ntn1 Gfap CKO mice display more symmetric forelimb movements than the controls in the exploratory reaching test. By Shapiro–Wilk test, the samples followed a normal distribution. Student’s t test, p < 0.05, *, N = 12 for control and N = 6 for CKO mice. F – H In the catwalk test, couplings describe the temporal relationship of two paws. The homologous coupling limb pair LF-RF is increased, whereas the values for limb pairs RF-LF were decreased in Ntn1 Gfap CKO mice compared with controls. By Shapiro–Wilk test, the samples followed a normal distribution. Student’s t test, p < 0.05, *, N = 7 for control and N = 6 for CKO mice. F Footprints of control and CKO mice and calculations (expressed as %) of coupling parameters. H Schematic representation for the pair LF-RF, in which LF acts as an Anchor Paw (yellow) and RF as a Target paw (blue).

Journal: Cell Death & Disease

Article Title: Ventricular Netrin-1 deficiency leads to defective pyramidal decussation and mirror movement in mice

doi: 10.1038/s41419-024-06719-1

Figure Lengend Snippet: A The body weight is comparable between control and CKO mice. By Shapiro–Wilk test, the samples followed a normal distribution. Student’s t test, p > 0.05, * N = 17 for control and N = 14 CKO mice. B , D No significant differences are found between the two groups in the traveled distance in the open-field ( B ), the time on the rods in the rotarod test ( C ) and the total time for mice landed to the floor in the pole test ( D ). E Ntn1 Gfap CKO mice display more symmetric forelimb movements than the controls in the exploratory reaching test. By Shapiro–Wilk test, the samples followed a normal distribution. Student’s t test, p < 0.05, *, N = 12 for control and N = 6 for CKO mice. F – H In the catwalk test, couplings describe the temporal relationship of two paws. The homologous coupling limb pair LF-RF is increased, whereas the values for limb pairs RF-LF were decreased in Ntn1 Gfap CKO mice compared with controls. By Shapiro–Wilk test, the samples followed a normal distribution. Student’s t test, p < 0.05, *, N = 7 for control and N = 6 for CKO mice. F Footprints of control and CKO mice and calculations (expressed as %) of coupling parameters. H Schematic representation for the pair LF-RF, in which LF acts as an Anchor Paw (yellow) and RF as a Target paw (blue).

Techniques:

A In embryonic hindbrain, Netrin-1 mRNA in the ventricular zone (VZ) is reduced in CKO mice, and accordingly, radial glial cell (green)-transported Netrin-1 protein (red dots) in the proximity of CST axons is decreased. B In adult CKO mice, the CST axons (blue) split into two bundles in the ventral hindbrain. The medial one crosses the midline, whereas the lateral one fails to do so and enters into the ipsilateral lateral funiculus of the spinal cord. 4 V 4th ventricle, df dorsal funiculus, ml midline, py pyramidal tract.

Journal: Cell Death & Disease

Article Title: Ventricular Netrin-1 deficiency leads to defective pyramidal decussation and mirror movement in mice

doi: 10.1038/s41419-024-06719-1

Figure Lengend Snippet: A In embryonic hindbrain, Netrin-1 mRNA in the ventricular zone (VZ) is reduced in CKO mice, and accordingly, radial glial cell (green)-transported Netrin-1 protein (red dots) in the proximity of CST axons is decreased. B In adult CKO mice, the CST axons (blue) split into two bundles in the ventral hindbrain. The medial one crosses the midline, whereas the lateral one fails to do so and enters into the ipsilateral lateral funiculus of the spinal cord. 4 V 4th ventricle, df dorsal funiculus, ml midline, py pyramidal tract.

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