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Celsr3 is required for Mauthner development. A,B) Tol-056 transgene expression of cytosolic GFP in Mauthner neurons in celsr3 sibling (A) and mutant (B) larvae. Mauthner axons are labeled with α-3A10 (neurofilament antibody) staining. All images and quantifications in figure are of 5 dpf larvae. Scale bar = 20μM, Z-projection. C) Mauthner cell number in celsr3 siblings (100% 2 M-cells; n = 51) and celsr3 mutants (12% 1 M-cell, 88% 2 M-cells; n = 67). D,E) Mauthner somas and axons in Tol-056 celsr3 sibling (D) and mutant (E). The Mauthner/spinal cord is pseudo-colored green for ease of visualization, while non-Mauthner/spinal cord GFP expression is magenta. Only z-frames with the Mauthner axon in focus are included. Arrows indicate where the Mauthner axons end and corresponding muscle segments, numbered anterior to posterior. Scale bar = 500μM, Z-projection. F,G) Magnified Mauthner axon ends in Tol-056 celsr3 sibling (F) and mutant (G). The top axon in (G) continues to segment 30. Scale bar = 50 μM, Z-projection. H) Mauthner axon lengths in celsr3 siblings and mutants measured by muscle segment containing the Mauthner axon end. Average sibling = segment 32 (n = 22 axons), mutant = segment 29 (n = 66 axons). I) Escape C1-bend ratio for celsr3 siblings and mutants that performed at least six fast escapes in response to 20 acoustic stimuli, normalized so all fish turning only one direction, left or right, appear at the same y position. n = 76 siblings, 6 mutants with one Mauthner (1M), 109 mutants with two Mauthners (2M). J) Escape frequency (Escape%) for individual Mauthners was calculated using total Escape% and turn bias ratio for individual fish; left C1-bend escape was assigned to right Mauthner, right C1-bend escape assigned to left Mauthner. n = 20 sibling Mauthners, 253 celsr3 mutant Mauthners. Points outlined in black indicate celsr3 mutant Mauthners from larvae with a single Mauthner (1M). Individual Mauthner escape% and axon length are not significantly correlated (simple linear regression, p = 0.63) in celsr3 mutants.
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