PMC

Muscle physiology in nrd^+/? and nrd^−/− larval zebrafish. (A) Averaged traces of 26–48 mEPCs shown with single (orange) and double (magenta) exponential fits for the decay component obtained from 72-hpf larval zebrafish. (B) Bar graphs quantifying the SSE for the single and double exponential fits of the decay component for the traces shown in A, wild-type (n= 6), nrd^+/? (n= 8) and nrd^−/− (n= 17). The SSE for a double exponential fit is significantly smaller for the nrd^−/− mutants (*P< 0.05, n= 17). (C) Bar graphs of single exponential fits for the decay component showing a significantly longer decay for the mEPCs acquired from nrd^−/− larval zebrafish.

Muscle morphology in nrd^+/? and nrd^−/− larval zebrafish. (A) Confocal images from 72-hpf nrd^+/? and nrd^−/− larvae labeled with the vital dye Draq 5 to reveal nuclei of fast muscle. Nuclei of fast muscle have a characteristic signature as they are ‘tilted’ at 45° (white dashed lines). (B) Cross-sections through muscle of 72-hpf nrd^+/? (left) and 72-hpf nrd^−/− larval zebrafish (right). Arrows in the nrd^+/? larva point to slow muscle fibers at the lateral aspect of the musculature. These slow fibers were not observed in nrd^−/− larval zebrafish. (C) Photomicrographs of 72-hpf nrd^+/? (left) and 72-hpf nrd^−/− larvae (middle) labeled with the antibody F59 to detect slow muscle fibers. The nrd^−/− larva lacks F59, slow muscle fiber labeling. Right, differential interference contrast image of the 72-hpf nrd^−/− larva. Scale bars, 20 μm.

Narrowminded mutants do not exhibit an increase in musculature twitch rates upon dechorionation. (A) Two 22-hpf embryos that were dechorionated are shown in the field of view. In these experiments, embryos were observed and videotaped for 30 min. We noticed in our initial experiments that some embryos lacked movements when dechorionated (embryo denoted by black arrow). (B) Behavioral examination quantifying the number of bends that occurred for each embryo for each minute. The embryos that lacked movement were separated from the rest of the group and along with the motile embryos, processed for immunohistochemistry. (C and D) The motile embryos possessed aat- or zn12-positive RB neurons arrows at 48 hpf. Those that did not exhibit any movements lacked RB neurons. Scale bars, 20 μm.

nrd^−/− embryos and secondary motoneuron axon abnormalities. The antibody zn5 was used to label secondary motoneuron axons in nrd^+/? and nrd^−/− embryos. (A) Left, cartoon depicting the pattern of zn5 labeling observed in 48–54-hpf embryos. At this stage of development, a prominent ventral–medial projection is apparent (arrow) but no dorsal projection is detected by zn5. A ventral–lateral projection is also present (open arrowhead). Right, photomicrographs of ∼50-hpf nrd^+/? and nrd^−/− embryos processed for zn5 immunohistochemistry. In the nrd^+/? embryo, arrows point to ventral axons that exit the spinal cord in a tight fascicle. In the nrd^−/− embryo, four of the five segments shown have axons that appear to be unraveling. The ventral projection labeled by the arrowhead appears normal. However, it appeared to have prematurely forked into two branches compared with those axons in the nrd^+/? embryo. (B) Photomicrographs of 54-hpf nrd^+/? and nrd^−/− embryos processed for zn5 immunohistochemistry. Arrows in the nrd^−/− embryo emphasize axons that exhibit pathfinding problems as extra branches are apparent. To the right, quantification indicated that almost all of the segments analyzed in nrd^+/? embryos had normal ventral axons (98 ± 2.0%, n= 52). In contrast, only 2 ± 1.7% of the segments analyzed (n= 61) in nrd^−/− embryos had normal ventral axons. Scale bar, 20 μm. *P< 0.05.

Pharmacological block of neural activity early in development phenocopies aspects of motoneuron morphology seen in nrd^−/− mutants. (A) Zebrafish embryos were exposed to varying concentrations of MS222 (tricaine) while in their chorions beginning at 15–17 hpf and then returning them to embryo medium at 30 hpf. The embryonic motor output (at 22 hpf) was reduced by 0.01–0.02% tricaine. (B) Photomicrographs of a 72-hpf control larva (top) and a 72-hpf tricaine-exposed larva (bottom). The antibody zn5 was used to label secondary motoneuron axons. The region denoted by the white circle in the bottom panel emphasizes a region where the dorsal projecting nerve appeared to stall, failing to extend into the dorsal myotome. (C) Photomicrographs of two tricaine-exposed zebrafish (60 and 72 hpf, respectively) emphasizing ventrally projecting axons. The antibody zn5 was used to label secondary motoneuron axons. In the 60-hpf embryo at the left, the region denoted by the white circle emphasizes a region where the ventral projecting secondary motoneuron axons appeared to stall before getting to the periphery. They then extended into the periphery appearing as a thin fiber (arrowheads). Ectopic branches (white arrows) are extending off the ventral–lateral projection. In the 72-hpf larva at the right, ectopic branches can be seen denoted by arrows. In these examples, the head is to the left. Scale bar, 20 μm.

nrd^−/− larval zebrafish have abnormal secondary motoneuron axon morphology. The antibody zn5 was used to label secondary motoneuron axons in nrd^+/? and nrd^−/− larvae. (A) Left, cartoon depicting the pattern of zn5 labeling observed in 72-hpf larvae. At this stage of development, a prominent ventral–lateral projecting nerve projection is present (open arrowhead) along with the ventral medial nerve (arrow) and dorsal nerve (arrowhead). Middle, zn5 labeling in a 72-hpf nrd^+/? larva and a 72-hpf nrd^−/− larva. Arrows point to dorsal axons which appear to be stunted and do not project to the periphery. The ventral axons also appear abnormal when compared with the control. To the right is a high-magnification photomicrograph of a ventral projecting secondary motoneuron nerve from a 72-hpf nrd^−/− larva. Note the extra branching. (B) Three-dimensional projections from the trunk regions of 72-hpf nrd^+/? and nrd^−/− larvae. As was the case for the single focal plane image shown in A, the dorsal projecting nerve in the nrd^−/− larva appeared to have stalled as it targeted to the periphery (white arrow). The white arrowhead points to a thin portion of the nerve where it appears that the axons resumed targeting to the periphery after being stalled. The ventral projection also exhibits ectopic branching. (C) High-magnification photomicrographs of ventral projecting secondary motoneuron axons from 144-hpf and 192-hpf nrd^−/− larval zebrafish. Black arrows point to ventral projecting motoneuron axons and not the processes associated with dorsal root ganglion neurons. Scale bars, 20 μm.

Behavioral characterization, motoneuron and muscle anatomy in nrd^−/− embryos. (A) Top, while still in their chorions, embryos were separated based on lack of movement. At 36 hpf, they were fixed and processed for aat immunohistochemistry. Middle, prior to 22 hpf, nrd^−/− embryos do not exhibit spontaneous twitches of the musculature. Ultimately, nrd^−/− embryos exhibit spontaneous twitches of the musculature at ∼24 hpf. Bottom, nrd^−/− embryos raised in embryo medium containing high [K⁺] (filled circles) exhibited a significant increase in bend rate compared with nrd^−/− embryos raised in normal embryo medium (open circles). (B) Top, the embryos that exhibited movement possessed RB neurons as shown by black arrows pointing to RB somata and black arrowheads pointing to the dorsal longitudinal fasciculus (DLF). The DLF partially comprises axons of RB neurons. Thus, these embryos were nrd^+/? siblings. Those that did not move lacked RB neurons and were nrd^−/− embryos. In the grey (inverted) panels, we also observed that the ventrally projecting motoneuron axons in embryos that lacked RB neurons (nrd^−/−) had abnormal branching patterns (black arrow) as shown in the very bottom panel. (C) Top, photomicrographs of 28-hpf nrd^+/? and nrd^−/− embryos labeled with the antibody F310, which detects fast muscle fibers. Bottom, photomicrographs of 28-hpf nrd^+/? and nrd^−/− embryos labeled with the antibody F59, which detects slow muscle fibers. (D) Photomicrographs of 28 hpf nrd^+/? and nrd^−/− embryos labeled with the antibody 4D9, which detects engrailed expressing muscle pioneers, show lack of engrailed expression in nrd^−/− embryos. Scale bars, 20 μm.

Motoneuron pathfinding errors in nrd^−/− embryos. The antibody znp1 was used to label primary motoneuron axons in nrd^+/? and nrd^−/− embryos. (A) Left, cartoon depicting the pattern of znp1 labeling observed in 22- to 48-hpf embryos. Initially, the ventral axon (CaP, arrows) projects ventrally and then turns back to the midline. The dorsal projecting axon (MiP, arrowhead) follows a similar trajectory to the dorsal periphery and then turns and projects to the midline. The dotted lines represent axonal trajectories to the midline that are in a different focal plane compared with the focal plane of the axons projecting to the distal periphery (solid lines). Right, znp1 labeling in a 30-hpf embryo showing the characteristic trajectories of primary motoneuron axons. The dotted line corresponds to the region of the choice point. When CaP axons make it to the choice point, they turn caudally and project into the periphery. (B) Photomicrographs of a 24-hpf nrd^+/? embryo (left) and a 24-hpf nrd^−/− embryo (right). Note that the axons in the nrd^−/− embryo do not exhibit the change in trajectory at the choice point like those axons in the nrd^+/? embryo. There are also ectopic branches projecting off these axons (arrows) in the mutant. (C) Photomicrographs of two nrd^−/− embryos, 22 hpf and 28 hpf. There are ectopic branches projecting off the CaP axons (arrows). (D) Photomicrographs of two 30 hpf nrd^−/− embryos. There are ectopic branches projecting off the ventral axons (arrows). (E) Photomicrographs of ventral motoneuron axons in nrd^+/? and nrd^−/− embryos. The motoneurons were revealed using znp1/zn1 immunohistochemistry. Using this procedure, somas of CaP and VaP (white arrowheads) were easy to detect as well as the ventral projecting axon. In the nrd^−/− embryo (right), the axon shown has an ectopic branch (white arrow), but only one motoneuron soma is present. It is likely a CaP motoneuron. In this example, the ectopic branching is not from the VaP motoneuron living longer in this segment. Scale bars, 20 μm.

Depolarizing activity early in development restores motoneuron morphology in nrd^−/− larvae. (A) Morphology of secondary motoneurons was analyzed in larvae exposed to high [K⁺] utilizing the antibody zn5. In 72-hpf, high [K⁺]-exposed nrd^−/− larvae, the dorsal axons project well into the periphery (compare with ). The ventral nerve projections appeared to have less axonal branches than nrd^−/− larvae not exposed to high [K⁺]. The percentage of dorsal projections that extended into the periphery was quantified to determine the extent of ‘restoration’ back to the normal phenotype caused by raising embryos in embryo medium containing high [K⁺]. In 72-hpf nrd^−/− larvae, 61 ± 5.0% of dorsal segments (n= 100) were innervated by a nerve compared with 100% in wild-type or sibling controls. In the high [K⁺] exposed nrd^−/− larvae, 100% of the dorsal segments analyzed (n= 65) were all innervated. In 72-hpf nrd^−/− larvae reared in high [K⁺] embryo medium after 27 hpf, 58 ± 7.2% of the dorsal segments analyzed (n= 61) were innervated by secondary motoneuron axons. (B) Quantification further revealed that primary motoneuron axonal development was influenced by depolarizing activity. In 22- to 26-hpf nrd^−/− embryos, 25 ± 5.5% of the CaP axons (n= 52) were normal. Abnormalities in the form of ectopic branching were observed in the remaining CaP axons. By 28–32 hpf, 41.0 ± 4.0% of the ventral projecting motoneuron axons were normal (n= 76). The remaining ventral axons exhibited ectopic branching or pathfinding errors. In contrast, ∼95% (103/108 total axons) of all the ventral motoneuron axons analyzed in 22- to 32-hpf nrd^+/? embryos were normal. In nrd^−/− embryos raised in high [K⁺] embryo medium, 68 ± 5.0% of the ventral primary motoneuron axons (n= 37) had a normal morphology. The remaining axons were still abnormal, exhibiting ectopic branching. Scale bar, 20 μm. *P< 0.05.