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