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Celsr3 is required for spiral fiber axon guidance and glial localization to the axon cap. A) The axon cap (dotted red box in circuit diagram) is comprised of excitatory spiral fiber axons (blue) and feedback inhibitory axons (pink) synapsing with the Mauthner (green) axon initial segment (AIS), surrounded by astrocyte-like glia (dotted line). B-E) A wild type larva with j1229a transgene expression of cytosolic GFP in Mauthner and spiral fiber neurons (B,C), hcrt:Gal4, UAS:Kaede/hcrt::Kaede transgene expression in a subset of spiral fibers (B,D; protein mostly converted from green to red), and α-3A10 staining (B,E). White brackets in (B) indicate spiral fiber somas. Yellow brackets in (D) indicate spiral fiber axons at the axon cap. White arrows in (E) indicate spiral fiber axons at midline crossing. All images and quantifications in figure are of 5 dpf larvae. Scale bar = 20μM, Z-projection. F-J) Magnification of box in (B) showing both Mauthner axon caps with TO-PRO-3 labeling nuclei (F,G), α-S100B labeling glia (F,H), Kaede labeling spiral fiber axons (F,I), and α-3A10 labeling Mauthner axons (F,J). Yellow arrowheads in (J) indicate Mauthner AISs. Scale bar = 20μM, single Z-frame. K,L) celsr3 mutant larva with Tol-056:GFP (K) and hcrt::Kaede (K,L) transgene expression. White asterisks indicate spiral fiber soma (note: only a subset of spiral fiber neurons are labeled due to variegated transgene expression). Yellow brackets indicate normal spiral fiber axon targeting to Mauthner axon cap. Red arrow indicates inappropriate spiral fiber axon targeting. Scale bar = 20μM, Z-projection. M,N) Mauthner axon cap region of sibling (M) and celsr3 mutant (N) Tol-056:GFP larvae. Kv1.1 localizes to Mauthner axon caps (yellow brackets) that spiral fiber axons target and accumulates at ectopic positions (red arrows) where spiral fiber axons frequently misproject. Scale bar = 20μM, Z-projection (dorsal range included for posterior Mauthner axon cap region and ventral range included for anterior spiral fiber axon misprojections, for clarity). O) Schematic for quantifying spiral fiber misprojections, measured as accumulation of Kv1.1 in ectopic “axon caps,” as in (N) (red arrows), within 60μM anterior to Mauthner AIS and 60μM lateral to midline (boundaries of spiral fiber soma). Left brain hemisphere is normal, with 0 ectopic caps, while right brain hemisphere has 1 ectopic spiral fiber cluster. For quantification in (P), this example would be counted as 0 (for left) and 1 (for right). P) Quantification of “ectopic caps,” presumed to represent spiral fiber misprojections, assessed by counting numbers of large Kv1.1 accumulation (>5μM diameter) rostroventral to position of normal axon cap, as in (O). For each larva, the left and right hindbrain were counted independently (two “hemispheres” per larva). In addition to ectopic Kv1 accumulation, all sibling and celsr3 mutant hemispheres with a Mauthner present had Kv1.1 accumulation at the endogenous axon cap region surrounding the Mauthner AIS. celsr3 mutant brain hemispheres where Mauthner neurons were absent only had ectopic caps. n = 10 sibling hemispheres, 58 celsr3 mutant hemispheres with Mauthners, 9 celsr3 mutant hemispheres without Mauthners. Q) Schematic for quantifying anterior-posterior position of ectopic caps quantified in (P). 1 = positioned at Mauthner AIS, 2 = <20μM from correct position, 3 = posterior to spiral fiber soma, 4 = at posterior spiral fiber midline crossing, 5 = at anterior spiral fiber midline crossing. For example, ectopic cap in (O) would be counted as 4. When the Mauthner neuron was absent, additional axon tracts labeled by Kv1.1 were used to identify where correct axon cap positioning was expected. R) Quantification of anterior-posterior position of ectopic caps from (P). For brain hemispheres with two ectopic caps, each position was counted separately. n = 63 celsr3 mutant ectopic caps in hemispheres with Mauthners, 17 celsr3 mutant ectopic caps in hemispheres without Mauthners. S-X) Sibling (S-U) and celsr3 mutant (V-X) larvae with Glast:myrGFP-p2A-H2AmCherry (membrane GFP, nuclear mCherry) transgene labeling astrocytes, as well as Tol-056:GFP and hcrt::Kaede (partially converted from green to red) transgenes. Green channel (myrGFP, GFP, and Kaede) in S,V,T,W; red channel (H2AmCherry, Kaede) in S,V,U,X. White asterisks indicate spiral fiber soma (note: only a subset of spiral fiber neurons are labeled due to variegated transgene expression). Yellow brackets indicate endogenous Mauthner axon cap, red brackets indicate ectopic localization of glia, red arrow indicates misprojected spiral fiber axons, and yellow arrowheads indicate two example glia localized correctly to the endogenous axon cap. Scale bar = 20μM, Z-projection. Y) Quantification of percent of sibling or celsr3 mutant larvae with zero, one (left or right), or two (left and right) anterior ectopic glial clusters. All larvae for quantification had two Mauthners. n = 16 siblings, 15 celsr3 mutants. Z) For larvae in (Y), glia on one side of the hindbrain (left or right hemisphere) were categorized as correctly localized to only the endogenous axon cap at the Mauthner AIS, localized to both the endogenous axon cap and an anterior ectopic location, localized only to an ectopic location, or not observed/missing. n = 32 sibling hemispheres, 30 celsr3 mutant hemispheres.
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