mouse dsred  (TaKaRa)

Journal: bioRxiv

Article Title: Tracking Satb2-positive retinal ganglion cells in zebrafish unveils developmental functional reorganization

doi: 10.64898/2025.12.25.696470

Figure Lengend Snippet: a) Two hypothetical models for the presence of multiple functional response types within the same retinorecipient tectal layer. Hypothesis 1: Multiple RGCs, each exhibiting a homogeneous response across all axonal branches, converge onto the same tectal layer. Hypothesis 2: A single RGC possesses distinct axonal branches with heterogeneous response profiles that terminate in a single tectal layer. b) Experimental workflow. Sparse labeling of Satb2-positive RGCs was achieved by injecting UAS(14x):GCaMP6s plasmid DNA into Tg(satb2:Gal4);Tg(UAS:mCherry) embryos at singe-cell stage. Functional calcium imaging was performed at 6-7 dpf, followed by whole-mount immunohistochemistry to match functional responses to terminal anatomy. c) Two-photon images of sparsely labeled terminals from individual RGCs at 7 dpf across two Z-planes (Z40, Z50). A, anterior; R, right. d) Post-hoc whole-mount immunostaining of the imaged cell in (c). GFP labels the injected GCaMP6s-expressing RGC; DsRed labels the three Satb2-positive recipient layers (SO, SFGS3, SFGS5). 3D reconstruction confirms that this neuron arborizes selectively within the SFGS3 lamina. Color indicates cluster assignment derived from (e). Scale bar: 50 µm. e) Functional response properties of the RGC shown in (c-d). Left: pixel-wise response heatmap (t-score) across 18 regressors from two imaging planes (Z40, Z50). Right: average response profiles shown as bar plot and normalized activity traces (the average of all pixels) (ΔF/F0), indicating strong preference for bog-dot stimuli, classified as EC5 (big dot). f-g) Same layout as (c-d) for another sparsely labeled RGC terminating within the SFGS5 layer. 3D reconstruction confirms laminar specificity. Color denotes functional cluster from (h). A, anterior; R, right. Scale bar, 50 µm. h) Functional response map of cell shown in (f-g). Left: pixel-wise t-score matrix for 18 regressors across two imaging planes. Right: average response profiles shown as bar plot and normalized activity traces (the average of all pixels) (ΔF/F0), showing strong activation to multiple motion-related stimuli, classified as EC7 (motion). i) Summary of reconstructed Satb2-positive RGC morphologies (N = 13) organized by laminar position. Each example represents one cell, color-coded by functional cluster identity. SO layer: EC2 (small dot). SFGS3 layer: EC3 (dimming/OFF), EC5 (big dot). SFGS5 layer: EC2 (small dot), EC5 (big dot), EC6 (looming), EC7 (motion).

Article Snippet: Then, whole mount larvae were first incubated in the primary antibody solution (chicken anti-GFP (A10262, Thermo Fisher Scientific/Invitrogen, 1:1000), mouse DsRed (632393, Clontech/Takara, 1:500), rabbit anti-Synapsin 1/2 (106002, Synaptic Systems, 1/1000)) for 7 days at 4°C, followed by the secondary antibody solution (goat anti-chicken Alexa Fluor 488 (A11039, Thermo Fisher Scientific/Invitrogen, 1:250), goat anti-mouse Alexa Fluor 555 (A21422, Thermo Fisher Scientific/Invitrogen, 1:250), goat anti-rabbit Alexa Fluor 647 (A21244, Thermo Fisher Scientific/Invitrogen, 1:250), DAPI (1:1000)) for 5 more days at 4°C.

Techniques: Functional Assay, Labeling, Plasmid Preparation, Imaging, Immunohistochemistry, Immunostaining, Injection, Expressing, Derivative Assay, Activity Assay, Activation Assay