PMC

dcc is required for counterbend directionality during touch-evoked startle responses. A, Schematic comparison of known neuronal and behavioral differences between head and tail touch-evoked startle responses (; ; ; ). B, Average frequency of touch-evoked counterbends correctly directed to the opposite side from the initial C-bend. Each larva was tested at 6–8 dpf with 10–15 tactile stimuli to the head and tail (n = 5 wild-type larvae, 12 dcc^tm272b larvae, 7 dcc^zm130198 larvae). *p = 0.0244 (two-tailed pairwise t test). **p = 0.0025 (two-tailed pairwise t test). C–F, Representative time series of 6 dpf wild-type (C, E, top) and dcc^tm272b mutant (C, E, bottom) larvae responding to tactile stimuli to the head (C, D) or tail (E, F). C, E, Panels include the points of maximal body curvature for the C-bend (“B1”) and counterbend (“B2”) of the startle responses. D, F, The total body curvatures of the larval responses depicted in C and E are graphed over 80 ms following initiation of the startle maneuver, with the wild-type response in blue and the dcc^tm272b mutant response in red.

dcc is required for commissural axonal projections of hindbrain interneurons, including the Mauthner/MiD2/MiD3 array. A–C, Confocal projections of hindbrain rhombomeres 4–6 of 60–70 hpf embryos stained with the antineurofilament antibody αRMO44 (black), from a dcc^tm272b/+ heterozygous sibling (A), a homozygous dcc^tm272b mutant (B), and a homozygous dcc^zm130198 mutant (C). A, B, The GFP enhancer trap transgene j1229a was also present to colabel the Mauthner array cell bodies with anti-GFP (red). Green asterisks indicate MiD3cl axons aberrantly extending laterally and/or rostrally. Yellow “×” indicates the cell body of an unscored T-reticular neuron extending a commissural axon through rhombomere 6 in panel C. White scale bars, 36 μm. For clarity, camera lucida tracings of the Mauthner arrays in these projections are presented in A′–C′. Mauthner axons are in blue (rhombomere 4) and Mauthner homolog axons are in red (MiD2cm pair from rhombomere 5, MiD3cl and MiD3cm pairs from rhombomere 6). The MiD3cl axon in C extends rostrally out of the presented image, then turns and extends ipsilaterally toward the posterior in a more lateral axon tract. D, Quantification of commissural versus ipsilateral axonal projections of hindbrain M-homolog neurons (MiD2cm, MiD3cm, MiD3cl) stained by αRMO44 for wild-type (+/+, n = 33 embryos), heterozygous (dcc^tm272b/+, n = 14 embryos), and dcc mutants (dcc^tm272b and dcc^zm130198, n = 24 and 13 embryos, respectively). The number of scored neurons is listed at the base of each bar. ****p < 0.0001. E, F, Confocal projections of hindbrain rhombomeres 4–7 of 6 dpf larval brains stained with an anti-neurofilament antibody (α3A10, red) and αGFP (green), from sibling (E) and dcc^tm272b mutants (F) carrying 2 copies of the j1229a GFP enhancer trap transgene. Blue arrowheads indicate discrete hindbrain commissure bundles labeled by α3A10.

The acoustic startle responses of dcc mutant larvae display exaggerated magnitude and disrupted counterbends. A, Homozygous dcc^ts239, dcc^tm272b, and dcc^zm130198 larvae show a significant increase in maximum head turning angle during short latency acoustic startle responses compared with their siblings. ****p < 0.0001 for each pair. Trans-heterozygous dcc^tm272b/dcc^zm130198 larvae show a similar increase in maximum head turning angle compared with wild-type siblings. ****p < 0.0001. dcc^tm272b/+ and dcc^zm130198/+ heterozygous larvae show no significant acoustic startle defects. Larvae received 20 acoustic stimuli each, and numbers of larvae analyzed per genotype are shown at the base of each column. B, C, Representative time series of 6 dpf wild-type (B, top) and dcc^tm272b mutant (B, bottom) zebrafish larvae responding to acoustic stimuli. B, Panels represent the points of maximal body curvature for the C-bend (“B1”) and counterbend (“B2”) of the acoustic startle response. The total body curvatures of the larval responses in B are graphed in C >80 ms after initiation of the acoustic stimulus. C, Black bracket represents the increase in maximal body curvature achieved during the C-bend of dcc mutants. D, The average frequency of counterbends performed following acoustically evoked C-bends by 6 dpf dcc^tm272b mutant larvae (dcc, n = 42 in red) and their wild-type siblings (WT, n = 31 in blue) across 20 identical acoustic stimuli. **p = 0.0054 (one-tailed t test with Welch's correction for unequal variances). Each point graphed represents a single larva. E, F, Tail curvature during a spontaneous swim maneuver performed by a 6 dpf wild-type sibling (E) and a dcc^tm272b mutant larva (F). Three repeated leftward tail bends by the mutant are highlighted with arrows in F. G, Frequency of spontaneous swim maneuvers with strictly left/right alternating tail bends for wild-type (n = 8 larvae) and dcc^tm272b mutants (n = 12 larvae). ****p < 0.0001. Each point represents a single larva.

The zebrafish dcc gene is mutated in spaced out. A, Recombination mapping placed spo^ts239 between a SNP marker in the tek gene (tie2, 6 of 1604 meioses) and an SSLP marker in the presumptive second intron of dcc (z23466, 1 of 1604 meioses). Although no mutations were observed in the dcc coding sequence of spo^ts239, a point mutation was detected in the spo^tm272b allele in the fourth Fibronectin Type III domain of DCC (FNIII, red ovals). The immunoglobulin-like domains (green horseshoes), transmembrane domain (black vertical bars), and cytoplasmic domains (conserved P1, P2, and P3 domains in blue) are all unperturbed in these alleles. B, The spo^tm272b allele carries a T-to-A mutation, producing an isoleucine-to-asparagine missense in the DCC protein sequence (I⁷⁹⁰ → N). C, The isoleucine residue disrupted in the spo^tm272b allele (I⁷⁹⁰) is in a highly conserved region of the fourth fibronectin domain of DCC. D–F, Binding of FLAG-Netrin to Cos-7 cells transfected with wild-type zebrafish dcc-egfp (D), dcc^tm272b-egfp (E), or musk-egfp (F). FLAG-Netrin signal alone for each is shown in D′–F′. G, Quantification of background-corrected total cell immunofluorescence of the FLAG epitope of GFP-positive transfected cells following FLAG-Netrin overlay. The number of cells analyzed per condition are shown at the base of each column. ****p < 0.0001, *ns, not significant. H, The dcc^zm130198 allele carries a 5.2 kb retrotransposon insertion in the 5′UTR of dcc, 106 nucleotides upstream of the start codon. I, Quantitative RT-PCR showed a significant decrease in dcc transcript levels in dcc^zm130198 homozygotes at 50 hpf (*p = 0.0342) and in dcc^ts239 homozygotes at 148 hpf (**p = 0.0015). ns, not significant. Four independent RNA samples were analyzed in each condition.

Ipsilaterally misprojecting MiD2/MiD3 neurons in dcc mutants result in counterbend directionality defects. A, B, Confocal projections of hindbrain rhombomeres 4–7 in a live 3-d-old larva carrying 2 copies of the j1229a GFP enhancer trap transgene in green, immediately before laser ablation of the MiD2/MiD3 homologs (A) and 1 h after ablation (B). The cell body positions of Mauthner homologs in rhombomeres 5 and 6 targeted for ablation are marked with magenta asterisks. Images are composites of multiple overlapping z-projections, registered using Mauthner axons and unablated cells. Projections were individually adjusted for brightness and contrast to permit consistent visibility of the Mauthner array and debris, and to confirm ablation of neurons rather than photobleaching. C, Wild-type siblings with bilateral MiD2/MiD3 ablation (light blue) and unablated controls (dark blue), as well as similarly ablated and unablated dcc mutants (pink and red, respectively) were tested at 6 dpf with 10 tactile stimuli to the head, and responses were specifically scored for counterbend directionality relative to the initial startle bend. Numbers of larvae analyzed per genotype are shown at the base of each column. All individuals carried 2 copies of the j1229a GFP transgene to visualize the Mauthner/MiD2/MiD3 array for ablation. **p = 0.0100. D, A model for the role of MiD2/MiD3 neurons in the dcc mutant counterbend phenotype. Head touch activates the Mauthner/MiD2/MiD3 hindbrain array through the trigeminal sensory neurons (black). This reticulospinal array activates trunk motor neurons (“MN” in green) to initiate the contralateral C-bend (“Bend 1”), as well as proposed commissural interneurons of the caudal hindbrain and/or spinal cord (“X” in orange), which directly or indirectly activate motor neurons on the opposite side for the subsequent counterbend (“Bend 2”). In wild-type larvae where the MiD2/MiD3 neurons have been ablated (top right panel, red dotted lines indicating ablated neurons), Mauthner activity alone is sufficient to activate trunk motor neurons for the contralateral C-bend and commissural “X” interneurons to allow an appropriate counterbend. In dcc mutants (bottom left), these commissural interneurons are bilaterally activated, which resolves in some responses to produce a counterbend on the same side as the C-bend. In dcc mutants where the MiD2/MiD3 neurons have been ablated (bottom right), this bilateral conflict is removed and appropriate counterbend direction is restored. Inactive neurons in each scenario are shaded gray.