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Figure 1.
touchdown mutants are hypopigmented and display a transient loss of touch-evoked behaviors. A, Lateral view of wild-type (left) and touchdown mutant (right) larvae at 55 hpf. Arrows point to a few of the visible melanophores, which contribute to pigmentation in wild-type zebrafish but are missing in touchdown larvae. Scale bar, 200 μm. B, Frames of videos showing touch-evoked escape contractions at 26 hpf of wild-type (top) and touchdown (bottom) embryos. 0 ms is the video frame preceding contact of the embryo by the stimulating probe. Scale bar, 500 μm. C, The percentage of wild-type (black) and touchdown mutants (red) displaying touch-evoked contractions between 19 and 92 hpf (n = 24 from three clutches) showing that touchdown mutants do not respond between 52 and 63 hpf. Values represent the average ± SEM. *p < 0.05, t test. D, Video frames showing touch-evoked swimming at 55 hpf of wild-type (top) but not of touchdown (bottom) larvae. Of note, swimming larvae sometimes appear in two places as the behavior was faster than the sampling rate of the camera. Scale bar, 500 μm. E, The percentage of wild-type (black) and touchdown mutants (red) displaying touch-evoked swimming between 19 and 92 hpf (n = 24 from three clutches).
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Figure 2.
touchdown alleles tdotz310c, tdob508, and tdomi174 harbor mutations that abolish channel activity. A, A graphic representation of TRPM7 indicating the location of mutations in the three alleles of touchdown. CC, Coiled-coil domain; STP, serine-threonine-proline rich domain; KD, kinase domain. B, Average current responses of oocytes expressing wild-type TRPM7 or touchdown alleles to voltage ramps between â100 and +60 mV (n = 10 oocytes for each allele) showing that all three alleles lack channel activity (i.e., the currents in response to ramps were not significantly different from those of uninjected oocytes). Values represent the average ± SEM. *p < 0.05, t test.
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Figure 3.
The touchdown phenotype results from the loss of TRPM7 during larval development. A, Histogram showing the percentage of embryos injected with the indicated antisense MO that responded to tactile stimuli with escape contractions at 24 hpf (n > 60 from three clutches). Injection of trpm7, trpm6, and trpm7+ trpm6 antisense MOs did not affect touch-evoked contractions. B, A phylogenetic tree showing the assignment of the identified protein as the zebrafish ortholog of TRPM6. C, Average current responses of oocytes expressing wild-type TRPM7 or TRPM6 to voltage ramps between â100 and +60 mV (n = 10 for each allele), showing that TRPM6 exhibits a outwardly rectifying current similar to TRPM7. *, Current values of TRPM6 and TRPM7 that were significantly different from uninjected controls (p < 0.05). The amplitude of TRMP6 responses at +60 mV was significantly smaller than TRPM7 responses.
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Figure 4.
Tactile stimuli in touchdown mutants fails to activate second-order neurons and the locomotor network that generates swimming in zebrafish. A, Schematic of a minimal touch-evoked circuit in zebrafish. MN, Motor neuron; SM, skeletal muscle; HB, hindbrain; SC, spinal cord. Dashed line designates the dorsal midline; anterior is up. B, Extracellular recordings from wild-type and touchdown mutant larva using electrodes positioned to detect Mauthner neuron spikes showing that tactile stimulation activates M cells in wild-type but not touchdown larvae at 55 hpf. Gray bars indicate time of the stimulus to the puffer pipette. C, Several minutes of NMDA-evoked fictive swimming episodes recorded from axial skeletal muscle in a wild-type larva (55 hpf) in the presence of 3 μM curare, a concentration sufficient to attenuate membrane depolarization below the level necessary to induce excitation–contraction coupling. D, A faster sweep of NMDA-evoked fictive swimming detailing episode period, episode duration, and swimming frequency (frequency of endplate potentials within an episode). The first episode lasted 1.06 s and had 22 peaks, for a swim frequency of 20.75 Hz. E, Cumulative plots of episode periods and durations in wild-type and touchdown mutant larvae showing that episode period and durations are not affected by the mutation. F, Histogram showing that the frequency of NMDA-evoked fictive swimming is comparable between wild-type and touchdown mutant larvae (n = 5 for each).
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Figure 5.
Selective restoration of TRPM7 expression in sensory neurons is sufficient to restore touch-evoked responses. A, Top, Lateral view of a 55 hpf larva labeled with a riboprobe against trpm7 showing that trpm7 is expressed widely in the larva. Boxed area indicates enlarged area shown below. Scale bar 200 μm. Bottom, Enlarged area highlighting a few RB sensory neurons expressing trpm7, with some RBs indicated with arrows. Scale bar, 50 μm. B, Top, Rescue plasmid comprised of the sensory neuron enhancer/promoter construct zCREST2-hsp70:TRPM7-eGFP. Bottom, Lateral view of the dorsal spinal cord following injection of the sensory enhancer/promoter construct labeled with anti-GFP showing that TRPM7-eGFP is expressed by many RBs, some of which are marked by arrows. Dashed line denotes the dorsal boundary of the spinal cord. Green labeling above dashed line was also observed in uninjected embryos, indicating that is background labeling of the skin by anti-GFP. Scale bar, 50 μm. C, Expression of TRPM7 selectively in RBs rescues touch-responsiveness in touchdown mutant larvae. Video frames of touch-evoked swimming from a 55 hpf hypopigmented touchdown mutant expressing the rescue plasmid. 0 ms is the video frame preceding contact of the embryo by the stimulating probe. Scale bar, 500 μm.
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Figure 6.
Restoration of touch-evoked responsiveness by expression of TRPM7 lacking kinase activity or with altered ion selectivity. A, Currentâvoltage relations of TRPM7 mutants expressed in Xenopus oocytes. Each point represents the average (±SEM) from 10 oocytes expressing the indicated construct minus the average from 10 uninjected oocytes at the same membrane potential. The membrane potential was changed by a slow ramp (3.3 mV/s) from â100 to +60 mV. Left, Response of a TRPM7 mutant lacking a residue necessary for kinase activity (TRPM7D1686A). Middle, Response of a TRPM7 mutant predicted to be pore killing (TRPM7E1026K). Right, Response of a TRPM7 mutant predicted to change the properties of the pore (TRPM7E1026Q). B, Currentâvoltage relations of wild-type or TRPM7E1026Q-injected oocytes in normal ORS containing 98 mM Na and 1.3 mM Mg, and a modified ORS with 8 mM Na and 60 mM Mg (60 Mg2+). C, Average ability of rescue plasmids harboring amino acid substitutions to restore touch-responsiveness in touchdown mutant larvae, which were identified by their hypopigmentation phenotype.
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Figure 7.
The gross morphology of RBs appears normal in touchdown mutants. Top, A lateral bright field view of the trunk and tail regions from ssx-mini-ICP:eGFP transgenic wild-type and touchdown mutant larvae at 55 hpf. Rostral is to the left and dorsal is up. Scale bar, 50 μm. Middle, Confocal microscope view of the same regions displaying RB cell bodies (detected by using anti-GFP and a fluorescent secondary antibody) superimposed over the bright field image. The microscope was focused at the level of the RB cell bodies. Bottom, The same larvae viewed in a different focal plane that highlights RB peripheral neurites within the skin.
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Figure 8.
Cutaneous mechanoreceptors respond to tactile stimuli in touchdown mutants. A, A representation of the preparation used to record electrical and tactile-evoked responses in RBs. The dashed line designates the dorsal midline. The light gray area indicates the region in which the skin and muscle were removed to allow access to a contralateral RB cell body with an intact peripheral neurite (white). The stimulating probe was placed on the skin directly overlying the peripheral neurite. B, RB responses to depolarizing current injections (2 ms) to the cell body, and extracellular electrical stimulation (1 ms) of the peripheral neurite showing that the RBs are excitable in wild-type and touchdown mutant larvae. C, Touchdown mutants possess both type I and type II RBs (see text). In type I RBs, a subthreshold tactile stimulus (black) applied to the peripheral neurite resulted in an observable generator potential (arrows) at the onset and offset of the stimulus. In these RBs, increasing the amplitude of stimuli eventually triggered action potentials (cyan) at the onset and the offset of the stimulus. In type II RBs, activation by tactile stimuli produced action potentials (cyan) without visible generator potentials (arrowheads) in response to subthreshold stimuli (black).
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Figure 9.
Elevation of extracellular divalent cations rescues touch responsiveness in touchdown mutants. A, Video images of 55 hpf touchdown mutant larvae in 5 mM Ca2+ showing no response to touch when the skin was not removed (top), and exhibiting swimming in response to touch when the skin over several rostral somites (â¼5â10) had been peeled, allowing rapid diffusion of elevated divalent cations into the CNS (bottom). 0 ms is the video frame preceding contact of the embryo by the stimulating probe. Scale bar, 1 mm. B, Average responsiveness of unpeeled touchdown mutant larvae in the presence of 5 mM Ca2+ to tactile stimuli compared with peeled mutant larvae in the presence of 5 mM Ca2+, Ba2+, Sr2+, or Mg2+ (n = 15 from three clutches). Ca2+, Sr2+, and, to a lesser extent, Ba2+, but not Mg2+, increased responsiveness to touch of peeled touchdown mutants. *p < 0.05, t test.
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