Journal: Biology Open

Article Title: Neuronal cell culture from transgenic zebrafish models of neurodegenerative disease

doi: 10.1242/bio.036475

Figure Lengend Snippet: Images of cultured 24 hpf Islet1:GFP zebrafish embryos stained with zebrafish-specific neuronal markers to confirm that the cell cultures contain various types of neurons. (A) An Islet1:GFP motor neuron within the cultures is positively stained (red) for the neuronal marker 39.4D5 (islet1 and islet2 homeobox). (B) Another Islet1:GFP motor neuron, and nearby islet1:GFP negative cells, are stained positively (red) for the neuronal cell surface marker Zn12, indicating the inclusion of other types of neurons in addition to motor neurons. Scale bar: 10 µm.

Article Snippet: Non-specific antibody binding was blocked by incubation by 5% goat serum (in PBS) prior to a 1 h incubation with one of the following primary antibodies: for islet 1 and islet 2 homeobox, 39.4D5 (1:50) and neuronal cell surface marker Zn12 (1:50), all obtained from the Developmental Studies Hybridoma Bank; for anti-polyglutamine (PolyQ) (Millipore, clone 5TF1-1C2|MAB1574).

Techniques: Cell Culture, Staining, Marker

Journal: bioRxiv

Article Title: Theory of branching morphogenesis by local interactions and global guidance

doi: 10.1101/2021.08.30.458198

Figure Lengend Snippet: Developmental stages of caudal fin innervation, schematic of the mCherry-based experimental strategy and in vivo caudal fin imaging. (A) Developmental stages of zebrafish caudal fin innervation. Top panel - 5 dpf zebrafish wholemount with red box indicating the Region of Interest (ROI): caudal fin. Several other features such as neuromasts and lateral line are visible. All tubulin positive fibers are stained with anti-acetylated tubulin antibody and presented in cyan and HuC positive cell bodies are presented in red. Different developmental stages are presented −24 hpf, 48 hpf and 5 dpf. First sensory innervation starts to appear in the caudal fin area at 24 hpf, developing into a dense network of innervation by 48 hpf (egg hatching) and reaches maximum density at 5 dpf (swimming behavior). HuC/D positive cell bodies and Tubulin positive neuronal projections are visible on all stages. Caudal fin outline is marked with the dashed lines. Magnified inset on 5 dpf stage reveals the puncta-like tubulin immunostaining which compromised potential 3D reconstruction of single neuron peripheral arborization. Indeed, a continuous signal would be preferable for reliable reconstructions, as well as sparse labelling of neurons, justifying our mCherry-based strategy. (B) Experimental scheme of our Mosaic transient transgenic strategy that lead to mCherry mosaic labelling of neurons in 5 dpf zebrafish. (1) Using GAL4-Upstream Activating Sequence (GAL4-UAS) methodology, we generated transient transgenic fish in which a sparse number of neurons will express mCherry. Injection of genetic vector carrying UAS-mCherry-caax construction to 1-4 cell stage fertilized egg of Tg(HUC:GAL4;UAS:synaptophysin-GFP) lead to mosaic UAS-mCherry caax incorporation in some cells, while in the transgenic fish line Tg(HUC:GAL4;UAS:synaptophysin-GFP) synaptophysin fused GFP is expressed at the presynaptic site in presumably all HUC expressing cells (arrowhead direction corresponds to transcription and translation processes). As a result, mCherry mosaic expression will be visible in cell membranes in occasional neurons. (2) During selection process only zebrafish with labelled neurons in the caudal fin were considered for further analysis. Selection was performed at 5 dpf to ensure developed branching of caudal fin neurons. Zebrafish with mCherry fluorescence in the caudal fin were manually collected and used for the analysis. (C) In vivo confocal imaging of 5 dfp mCherry-caax-XHUC:GAL4:synaptophysin-GFP zebrafish caudal fin. Magnified inset shows lack of punctiform artifacts. This feature makes our mCherry reporter line preferable for neuronal branching reconstruction and thus was used in all experiments to visualize generation of neuronal trees.

Article Snippet: The primary antibodies utilized were anti-acetylated tubulin (Neuronal marker, Gene Tex), anti-HuC/HuD neuronal protein and (Abeam), all diluted 1/800 in blocking solution.

Techniques: In Vivo, Imaging, Staining, Immunostaining, Transgenic Assay, Sequencing, Generated, Injection, Plasmid Preparation, Expressing, Selection, Fluorescence

Journal: bioRxiv

Article Title: Sensory neurons sprouting is dependent on osteoclast-derived extracellular vesicles involving the activation of epidermal growth factor receptors

doi: 10.1101/259218

Figure Lengend Snippet: A. Representative images of DRG outgrowth after treatment (stained for βIII-tubulin, scale bar 500 μm). Neurobasal supplemented with nerve growth factor (NB); bone marrow stromal cells conditioned medium (BMSC); Pre-osteoclast conditioned media (Pre-OC), and mature osteoclast conditioned media (OC) was collected and added to embryonic DRG culture. B. Automatic axonal outgrowth quantification of DRG. Data represented as a violin plot. Non-conditioned osteoclast medium (OCm) represented by the dashed line. Statistical analysis was performed vs. controls – NB and OCm (*) and between groups (#). C. Analysis of secretome neurotrophins. Quantification of NGF, BDNF, NT-3, and NT-4/5 by ELISA on BMSC, Pre-OC and mature OC conditioned media. Data represented as mean±SD.

Article Snippet: DRG cultures were incubated with an antibody directed against the neuronal-specific marker βIII tubulin (Promega, Madison, WI, United States) diluted 1:2000 in blocking solution, overnight at 4°C.

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Sensory neurons sprouting is dependent on osteoclast-derived extracellular vesicles involving the activation of epidermal growth factor receptors

doi: 10.1101/259218

Figure Lengend Snippet: A. Quantification of axonal outgrowth of sensory neurons blocked with epidermal growth factor receptor (EGFR) inhibitor – Erlotinib. B. Representative images of DRG treated with different concentrations of Erlotinib (βIII tubulin in green and nuclei in blue, scale bar 500 μm). C. Quantification of axonal outgrowth of sensory neurons blocked with ErbB2 receptor inhibitor – Trastuzumab. D. Representative images of DRG treated with different concentrations of Trastuzumab (βIII tubulin in green and nuclei in blue, scale bar 500 μm). Data represented as box & whiskers (centerline is plotted at the median. Whiskers represent minimum to maximum range) *p≤0.05.

Article Snippet: DRG cultures were incubated with an antibody directed against the neuronal-specific marker βIII tubulin (Promega, Madison, WI, United States) diluted 1:2000 in blocking solution, overnight at 4°C.

Techniques:

Journal: bioRxiv

Article Title: Sensory neurons sprouting is dependent on osteoclast-derived extracellular vesicles involving the activation of epidermal growth factor receptors

doi: 10.1101/259218

Figure Lengend Snippet: A. Quantification of axonal sprouting of DRG stimulated with osteoclast secretome and osteoclast secretome with the functional anti-β1 integrin. Data represented as box & whiskers (median, whiskers represent minimum to maximum range), ****p≤0.0001. B. Representative images of DRG treated with osteoclast secretome and osteoclast secretome with the functional anti-β1 integrin. Staining for βIII tubulin in green and nuclei in blue, scale bar 500 μm. C. Levels of EGFR family phosphorylation assessed by RTK array. Values normalized by the neurobasal control. D. Representative images of EGFR phosphorylation by immunocytochemistry after treatment with NGF-supplemented neurobasal, osteoclast secretome, osteoclast secretome with the functional anti-β1 integrin and osteoclast secretome with anti-β1 integrin isotype. Phosphorylated EGFR (pEGFR) in red, βIII tubulin in green and nuclei in blue (left column); zoom in images of the isolated channel for the pEGFR (right column). Scale bar 100 μm.

Article Snippet: DRG cultures were incubated with an antibody directed against the neuronal-specific marker βIII tubulin (Promega, Madison, WI, United States) diluted 1:2000 in blocking solution, overnight at 4°C.

Techniques: Functional Assay, Staining, Immunocytochemistry, Isolation

Journal: bioRxiv

Article Title: Sensory neurons sprouting is dependent on osteoclast-derived extracellular vesicles involving the activation of epidermal growth factor receptors

doi: 10.1101/259218

Figure Lengend Snippet: A. Characterization of osteoclasts-derived EV by Western blot using CD81 membrane marker (EV vs. Supernatant (SN) EV depleted). B. Transmission electron microscopy of osteoclasts-derived EV (white arrows) by negative staining (100 000x). C. Representative images of DRG treated with osteoclast secretome and EV-depleted osteoclast secretome. Staining for βIII tubulin in green and nuclei in blue, scale bar 500 μm. D. Quantification of axonal sprouting of DRG stimulated with osteoclast secretome vs. osteoclast secretome lacking the EV. Data represented as box & whiskers (median, whiskers represent minimum to maximum range), *p≤0.05. E. Levels of RTK phosphorylation assessed by RTK array. Values normalized to the neurobasal control. F. Network analysis of osteoclast-derived EV enriched fraction according to the cellular components. Protein network (edge-weighted spring embedded layout) representing the most significant clusters associated with the identified proteins. Gene ontology (GO) term fusion was applied. Pathways with p value lower than 0.01 were considered. Tree interval was applied from 1 to 2 level. The size of the nodes is indicative of the number of associated proteins. The increase in red color gradient represents higher statistical significance. High resolution networks provided as supplemental material.

Article Snippet: DRG cultures were incubated with an antibody directed against the neuronal-specific marker βIII tubulin (Promega, Madison, WI, United States) diluted 1:2000 in blocking solution, overnight at 4°C.

Techniques: Derivative Assay, Western Blot, Marker, Transmission Assay, Electron Microscopy, Negative Staining, Staining