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The zebrafish space cadet gene controls axonal pathfinding of neurons that modulate fast turning movements

¹ Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6058, USA
² Department of Neurobiology and Behavior, Life Science Building, State University of New York at Stony Brook, New York, NY, USA

View larger version (142K): [in a new window]   Fig. 1. Mutant space cadet larvae exhibit specific swimming defects. Individual frames from high-speed videos showing (A) a stimulus induced escape response in wild-type larvae, (B) stimulus induced double turns in space cadet larvae and (C) spontaneous multiple turns in space cadet larvae. (A) Upon stimulation, the wild-type larva first bends its body and tail into a C-like profile, resulting in a fast displacement of the head away from the source of the stimulus (A1-A4). The C-like bend is first followed by a counter turn (A5-A6). Then, the larva rapidly accelerates away from its initial position (A7-A12). (B) space cadet larva responding to tactile stimulation with two successive large turns in the same direction (first turn B1-B6, second turn B7-B9). (C) A space cadet larva performing multiple, successive turns in the absence of tactile stimulation, resulting in a rotating movement (first turn C1-C6, second turn C7-C9 and third turn C10-C12)

View larger version (66K): [in a new window]   Fig. 2. In space cadet mutants, a subset of hindbrain commissures and the Mauthner axon cap are affected. Low magnification, dorsal views of the head of 120 hpf wild-type (A) and space cadet larvae (B) stained with the neurofilament antibody 3A10. All major trajectories appear unaffected in space cadet mutants. (C) In wild-type larvae, higher magnification reveals two prominent commissures in rhombomere 3 (arrows), the paired Mauthner cells (labeled M) with the characteristic axon cap at the initial segment of the axon (arrowheads) and a series of seven ladder-like commissural tracts in the caudal hindbrain (labeled 1-7). (D) In space cadet mutants the two prominent commissures in rhombomere 3 are strongly diminished or absent and the axon cap structure is not detectable. The ladder-like commissures in the caudal hindbrain appear less organized (black and white asterisks) and the caudal most commissure is missing in 35% of the space cadet larvae (red arrow). (E) High magnification view of the two rhombomere 3 commissures (arrows) and the Mauthner soma with the surrounding axon cap (arrowheads). (F) In space cadet mutants both commissures and the axon cap are strongly reduced. Dorsal view on the hindbrain of wild-type (G) and space cadet larvae (H), in which reticulospinal neurons (arrows) were retrogradely labeled (see also Table 1). Similarly, analysis of commissures in the midbrain revealed no difference between wild-type (I) and space cadet mutant larvae (J).

View larger version (165K): [in a new window]   Fig. 3. In space cadet mutants the two rhombomere 3 commissures and the Mauthner axon cap fail to develop. Developmental analysis of 3A10-labeled axonal tracts in the hindbrain of wild-type (A,C,E) and space cadet larvae (B,D,F). (A,B) At 72 hpf, (C,D) 96 hpf and (E,F) 120 hpf. In wild-type larvae the two rhombomere 3 commissures start forming around 72 hpf (arrows in A), and their thickness increases over the next 48 hours (arrows in C,E). The Mauthner axon cap becomes first detectable around 96 hpf (arrowheads in C) and is readily visible at 120 hpf (arrowheads in E). In space cadet mutants (B,D,F) the two commissures and the Mauthner axon cap fail to form. Note that both structures, the two commissures and the Mauthner axon cap, develop around the same time, and that this time point also coincides with the onset of the space cadet swimming phenotype. The arrows point to the commissures, the arrowheads to the Mauthner axon cap.

View larger version (111K): [in a new window]   Fig. 4. space cadet swimming can be phenocopied by lesioning two rhombomere 3 commissures. Dorsal views of the head of 120 hpf larvae stained with a neurofilament antibody. A tungsten needle was used to separate the nervous system along the midline, the red line indicates the rostrocaudal extent of the cut. The swimming pattern of the operated larvae was analyzed 2 to 16 hours later. Severing midbrain commissures (A) produces mainly larvae with wild-type swimming, while severing the hindbrain along its entire length (B) phenocopies the space cadet swimming defect. Further lesions were restricted to different hindbrain regions, encompassing only the two commissures in rhombomere 3 (red line in C), and between the two rhombomere 3 commissures and the Mauthner neuron (green line in C). (D) Non- operated control larva, the arrows pointing to the rhombomere 3 commissures. (E) Larva in which only these two commissures but not the adjacent axons of the Mauthner neuron (M) were severed.

View larger version (65K): [in a new window]   Fig. 5. Trans-synaptic labeling of Mauthner neurons reveals connectivity defects with space cadet presynaptic spiral fiber neurons. (A) The calcium indicator calcium green dextran was injected into the ventral spinal cord to label Mauthner neurons. (B) In larvae with retrogradely labeled Mauthner neurons, the right Mauthner cell was injected with neurobiotin. In most larvae, the injected Mauthner cell soma died within 2-18 hours of neurobiotin injection, presumably owing to phototoxicity effects when CGD loaded neurons were exposed to intense light during neurobiotin injection. Therefore, the injected Mauthner cell soma is not visible. Deliberate injection of neurobiotin adjacent to the Mauthner cell did not label any neurons away from the injection site. (C-E) Wild-type larvae labeled with neurobiotin (C,E) or neurobiotin and 3A10 (D). (C) Injection into one wild-type Mauthner cell (M, outlined) labeled distinct sets of ipsilateral and contralateral neurons, most notably commissural spiral fiber neurons (arrows). (D) 3A10 antibody staining confirming that the two rhombomere 3 commissures contain spiral fiber axons. (E) High magnification view focused on spiral fiber cell bodies and the two commissures. (F-H) In space cadet larvae, neurobiotin injection (F,H at high magnification) revealed a similar distribution of labeled neurons located at the same level or caudally to the Mauthner cells. In contrast, neurobiotin labeled spiral fiber neurons and axons, as well as 3A10-labeled commissures are absent, demonstrating that in space cadet larvae spiral fibers are not connected to their synaptic target, the Mauthner cell.

View larger version (71K): [in a new window]   Fig. 6. space cadet mutant larvae display retinal ganglion cell (RGC) axons pathfinding defects. The RGC layer in wild-type and space cadet larvae was injected with DiI to visualize RGC axon trajectories. (A) All wild-type RGC axons crossed the midline (broken line) and projected to the contralateral optic tectum (cot). (B) space cadet RGC axons projected aberrantly to the ipsilateral optic tectum (iot) or stalled around the midline (arrow, C). (D,E) Histological sections of 120 hpf wild-type (D) and space cadet (E) larvae show that retinal organization as well as cellular morphology are unaffected in space cadet mutants (le, lens; inl, inner nuclear layer; ipl, inner plexiform layer; on, optic nerve; pe, pigment epithelium; prl, photoreceptor cell layer; rgc, retinal ganglion cell layer. Immunostaining of wild-type (F) and space cadet larvae (G) with zn-5 antibody at 50 hpf. The zn-5 antiserum stains the RGC somata and axons. In a small fraction of space cadet mutants, the optic nerve, after exiting the eye (arrow), is markedly thinner when compared with wild-type eyes.

View larger version (19K): [in a new window]   Fig. 7. A model for the function of spiral fiber neurons in wild-type and space cadet larvae. For simplicity, auditory input, spiral fiber neurons, commissural (yellow) and collateral PHP neurons (blue) are only shown on the left side. The broken line designates the midline. (A) The behavioral threshold of the Mauthner cell (M, red) is determined by the relation between excitation (sensory input, black) and inhibition (PHP neurons, blue and yellow). In wild-type fish, sensory inputs (black), e.g. from the vestibular system through the VIIIth nerve activate the Mauthner cell and PHP neurons, which in turn inhibit the Mauthner cell. At weak stimulus strengths, PHP-mediated inhibition dominates, but at higher sensory input strength, this inhibition is overcome (Faber and Korn, 1978). Spiral fiber neurons (green) are thought to produce excitatory stimuli within the Mauthner axon cap and might also activate inhibitory PHP neurons. Our results suggest that spiral fiber neurons synapse on additional hindbrain neurons. The inputs on the spiral fiber neurons are unknown. (B) In space cadet mutants, spiral fiber neurons fail to develop their normal commissural axonal trajectories and might project ipsilaterally. Absence of correct spiral fiber trajectories causes (1) reduced and (2) spurious activation of the escape response. (1) Lack of spiral fiber-mediated activation of the Mauthner cell (at the axon cap) might reduce the excitability of the Mauthner cell. (2) The absence of spiral fiber input on PHP neurons might reduce their inhibition of the Mauthner cell. As a consequence, weak (sensory) stimuli can overcome PHP-mediated inhibition, resulting in spurious activation of the escape response.