α2-Chimaerin regulates a key axon guidance transition during development of the oculomotor projection

Abstract

The ocular motor system consists of three nerves which innervate six muscles to control eye movements. In humans, defective development of this system leads to eye movement disorders, such as Duane Retraction Syndrome, which can result from mutations in the α2-chimaerin signaling molecule. We have used the zebrafish to model the role of α2-chimaerin during development of the ocular motor system. We first mapped ocular motor spatiotemporal development, which occurs between 24 and 72 h postfertilization (hpf), with the oculomotor nerve following an invariant sequence of growth and branching to its muscle targets. We identified 52 hpf as a key axon guidance "transition," when oculomotor axons reach the orbit and select their muscle targets. Live imaging and quantitation showed that, at 52 hpf, axons undergo a switch in behavior, with striking changes in the dynamics of filopodia. We tested the role of α2-chimaerin in this guidance process and found that axons expressing gain-of-function α2-chimaerin isoforms failed to undergo the 52 hpf transition in filopodial dynamics, leading to axon stalling. α2-chimaerin loss of function led to ecotopic and misguided branching and hypoplasia of oculomotor axons; embryos had defective eye movements as measured by the optokinetic reflex. Manipulation of chimaerin signaling in oculomotor neurons in vitro led to changes in microtubule stability. These findings demonstrate that a correct level of α2-chimaerin signaling is required for key oculomotor axon guidance decisions, and provide a zebrafish model for Duane Retraction Syndrome.

Figure 1.

Early development of the ocular motor system. Multiphoton lateral views of live Isl1:GFP (A, B, D, E, G, H, J), α-act:RFP (M), and Isl1:GFP × α-act:RFP (N) embryos or wt embryos immunostained for α-tubulin (K) at indicated stages. nIII, oculomotor nucleus; nIV, trochlear nucleus; nV, trigeminal nucleus. Schematic diagrams of ocular motor development at 30 hpf (C), 44 hpf (F), 48 hpf (I), and 54 hpf (L). O, Schematic of the zebrafish head at 48 hpf, showing the OMN in blue, the area imaged (blue square), and the orientation. D, Dorsal; R, rostral; te, tectum; hb, hindbrain; ov, otic vesicle. Blue, green, and red represent the OMN, trochlear, and abducens nerves, respectively; gray represents muscle progenitors; orange represents myosin-positive muscles. All panels are oriented with rostral left and dorsal to the top. A, Dotted line indicates edge of neural tube. H, Dotted line indicates putative subdivision between oculomotor dorsal subnuclei. Dotted circles represent ciliary ganglion. K, *Branch of trigeminal nerve. Yellow and red arrows indicate nerve branches to the MR and IR muscles, respectively; blue arrow indicates the nerve branch toward the IO muscle. Scale bars, 50 μm.

Figure 2.

Later stages of development of the ocular motor system. Multiphoton imaging lateral views of wt embryos immunostained for α-tubulin (A, D), presynaptic vesicles (SV2) (G, I), or acetycholine receptors (αBgt) (J) at indicated stages. K, Merged image of both I and J (red circle in these images represents background staining from retina). Montage of live imaging of Isl1-GFP with α-tubulin and myosin heavy chain (myHC) immunostaining to display the pathway of the OMN and trochlear nerve at 72 hpf (C). Multiphoton ventral view of a wt embryo immunostained with Zn-5 antibody (F). r5,6, Rhombomere 5,6. Live multiphoton lateral view of a α-actin:RFP embryo at 72 hpf (H). Yellow and red arrows indicate nerve branches to the MR and IR muscles, respectively; blue arrow indicates the nerve branch toward the IO muscle; white arrow indicates the nerve branch to the SR muscle. A, B, *Branch of the trigeminal nerve. F, G, Arrow and arrowhead indicate abducens nerve. Schematics of ocular motor development at 66 hpf (B) and 72 hpf (E) displayed as in Figure 1. All panels, except F, are oriented with rostral left and dorsal to the top, as in Figure 1. Scale bars, 50 μm.

Figure 3.

Imaging of single OMN neurons. Live-imaging of single neurons within the OMN expressing GFP before (A) or after (B) the transition point. Arrowheads indicate filopodia. Live-imaging (C) and fluorescently-extracted (D) images of neurons within the OMN producing distinct branches at 63 hpf. Colored arrows indicate distinct neural projections. Single neurons expressing either GFP, WT-α2-chn-wt or the G228S-α2-chn mutant form before (E) or after (F) the 52 hpf transition point. The yellow arrow indicates branches growing into the muscle area. *Branching/stalling point. Arrowhead indicates a neural projection heading dorsally from the branching point. Schematic representation of the phenotype observed in these embryos after the transition point (G) with stalling in G228S-α2-chn-expressing neurons. Dotted lines indicate projections heading dorsally in some cases. All panels are oriented with rostral left and dorsal to the top. Scale bars, 25 μm.

Figure 4.

Filopodial dynamics during the 52 hpf transition. A, Illustrative image of a single GFP-expressing neuron within the OMN contacting the MR/IR progenitors in the alpha-actin-RFP line. B, Filopodial appearance rate in single neurons expressing either GFP, WT-α2-chn or the G228S-α2-chn mutant form both before and after the 52 hpf transition. *p <0.001. n = 9, n = 4, and n = 5 neurons, respectively, for 49 hpf. n = 10, n = 8, and n = 6 neurons, respectively, for 54 hpf. There is no significant difference between the rate of filopodial appearance in G228S-α2-chn-expressing neurons compared with GFP-expressing neurons either at 49 or 54 hpf (p = 0.869). C–E, Polar plots and radar plots showing the filopodial distribution before and after the 52 hpf transition in neurons expressing constructs as indicated. C, GFP-expressing neurons; the distribution at 49 and 54 hpf is different (p < 0.001). n = 173 and n = 93 filopodia, respectively. D, WT-α2-chn-expressing neurons; the distribution is different at 49 and 54 hpf (p = 0.037). n = 89 and n = 70 filopodia, respectively. The filopodial distributions for GFP and WT-α2-chn-expressing neurons are not significantly different either before or after (p = 0.417 and p = 0.445, respectively). E, G228S-α2-chn-expressing neurons; the distributions are similar (p = 0.985). n = 128 and n = 89 filopodia for before and after, respectively. The filopodial distributions for G228S-α2-chn-expressing neurons at 49 hpf or 54 hpf are not significantly different from those of either GFP or WT-α2-chn-expressing neurons at 49 hpf (p = 0.513 and p = 0.718, respectively).

Figure 5.

OMN phenotypes observed after morpholino microinjection. Multiphoton lateral views of MO1-injected (A, D, G, J) or MO2-injected (B, E, H) morphant embryos immunostained for α-tubulin at 72 hpf. Phenotypes observed are labeled to left, including ectopic branches (A, B), misguided branches (D, E), hypoplasia of the OMN or missing branches (G, H), or stalling of the whole OMN projection (J). Arrows indicate ectopic nerve branches; arrowheads indicate hypoplastic or truncated nerve projections. *Branch of the trigeminal nerve. OMN schematics (C, F, I, L) are as in Figure 1, with misguided and ectopic branches shown in dark green. K, RT-PCR of total RNAs extracted from 48 hpf morphant embryos. For both wt and MO1-microinjected embryos, both the presplicing and mature forms of mRNA are detected after amplification with both exon and intron primers. After MO2 microinjection, only the presplicing mRNA is detected. M, N, Multiphoton lateral views of MO1-injected embryos and MO1 mismatch controls immunostained for α-tubulin and myHC (M) or α-tubulin and presynaptic vesicles (N) at 72 hpf. M, Arrowheads indicate multiple nerve branches growing into the IO muscle. N, Arrowheads indicate synaptic staining. O, Multiphoton lateral view of a control “mismatch MO1”-injected embryo immunostained for α-tubulin at 72 hpf. Yellow and red arrows indicate nerve branches to the MR and IR muscles, respectively; blue arrow indicates the nerve branch toward the IO muscle; white arrow indicates the nerve branch to the SR muscle. P–R, Control images of wt embryos immunostained as M–O. Scale bars, 50 μm.

Figure 6.

Cytoskeletal dynamics and α2-chn. Oculomotor growth cones stained for both stable (green) and unstable (red) tubulin transfected with a control scrambled shRNA (A) or with an α2-chn shRNA (B). Ratio of stable over unstable microtubule area in the distal 20 μm of these growth cones in both conditions (C). n = 39 and n = 71 neurons for control and shRNA, respectively. *p = 0.004.