19 research outputs found

    Generation and characterization of Kctd15 mutations in zebrafish

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    <div><p>Potassium channel tetramerization domain containing 15 (Kctd15) was previously found to have a role in early neural crest (NC) patterning, specifically delimiting the region where NC markers are expressed via repression of transcription factor AP-2a and inhibition of Wnt signaling. We used transcription activator-like effector nucleases (TALENs) to generate null mutations in zebrafish <i>kctd15a</i> and <i>kctd15</i>b paralogs to study the in vivo role of Kctd15. We found that while deletions producing frame-shift mutations in each paralog showed no apparent phenotype, <i>kctd15a/b</i> double mutant zebrafish are smaller in size and show several phenotypes including some affecting the NC, such as expansion of the early NC domain, increased pigmentation, and craniofacial defects. Both melanophore and xanthophore pigment cell numbers and early markers are up-regulated in the double mutants. While we find no embryonic craniofacial defects, adult mutants have a deformed maxillary segment and missing barbels. By confocal imaging of mutant larval brains we found that the torus lateralis (TLa), a region implicated in gustatory networks in other fish, is absent. Ablation of this brain tissue in wild type larvae mimics some aspects of the mutant growth phenotype. Thus <i>kctd15</i> mutants show deficits in the development of both neural crest derivatives, and specific regions within the central nervous system, leading to a strong reduction in normal growth rates.</p></div

    Activity of reticulospinal neurons in ethanol.

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    <p>Fluorescence intensity of Mauthner neurons (A–C), vestibulospinal neurons (D, F) and MiD3 neurons (E, F). Sound/vibration stimuli were applied to sectioned (A, D, E) or intact larvae (B) bathed in 0, 30, 100, 300 and 1000 mM ethanol. The black, blue, green, orange and red lines correspond to 0, 30, 100, 300, and 1000 mM ethanol, respectively. The peak of ΔF/F normalized to values at 0 mM was plotted against the EtOH concentration in C and F. (*P<0.05, **P<0.005, ***P<0.001 versus 0 mM ethanol). Six Mauthner neurons, eight vestibulospinal neurons, and five MiD3 neurons were measured. Stimuli were applied and calcium transients were elicited at each concentration 2–5 times. The numbers of samplings are shown in parentheses.</p

    The effects of ethanol on acoustic startle responses.

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    <p>Histograms showing the latency distribution of startle responses of free-swimming larvae after 1 hr exposure to 0 (A) and 300 mM ethanol (B). N in parenthesis is the number of responses analyzed (from a total of 100–120 larvae each). Percentage of larvae showing responses with latencies <15 ms (C) and >15 ms (D) after exposure to 0, 30, 100 and 300 mM ethanol. (* P<0.001 t-test compared to 0 mM group). N = 4 groups of 20 larvae (0, 30, 100 mM treatments) or 8 groups of 20 larvae (300 mM treatment).</p

    The effects of ethanol on spontaneous locomotion.

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    <p>Spontaneous locomotion kinematics of larval zebrafish (n = 20–25) after exposure to 0, 30, 100 and 300 mM ethanol for 30 min. Speed, distance, tail beat frequency, and swim amplitude are shown in A, B, C, and D, respectively. Numbers of analyzed larvae are shown in brackets (***P<0.001 versus 0 mM ethanol).</p

    Recovery and adaptation of neuronal activity in 300

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    <p> <b>mM ethanol.</b> Fluorescence intensities of Mauthner neurons and MiD3 neurons were measured in response to sound/vibration stimuli. Sectioned larvae were either washed after 30 min incubation of 300 mM ethanol (A) or bathed continuously in 300 mM ethanol (D, E). Black, red, green and blue traces correspond to 0, 30, 60 and 90 min, respectively. The peaks of ΔF/F normalized to 0 min were plotted against time in B (for A) and F (for D, E) (*P<0.05, ***P<0.001 versus 0 min). Five Mauthner neurons and six MiD3 neurons were measured. Stimuli were applied and calcium transients were elicited at each concentration 2–5 times. The numbers of samplings are shown in parentheses. (C) AO staining in 0 mM (top) and 300 mM (bottom) ethanol are shown. Apoptosis in olfactory organs is marked with arrowheads. Apoptosis of neuromasts, located on the body surface, are shown with arrowheads in insets. Nonspecific signals are observed in the gut (Arrows).</p

    <i>Kctd15</i> mutants are smaller in size.

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    <p>A) Growth of wild-type and mutant larvae, measured at 5, 12, 18, 22 and 28 dpf. While the difference in size is noticeable at 12 dpf (B), the difference is not significant until 18 dpf (C), when a range of sizes is seen, ranging from somewhat smaller (mut #1) to much smaller (mut #2). Adult mutants remain significantly smaller than wild-type siblings, even after a year (D,E).</p

    <i>kctd15</i> mutants have more melanophores.

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    <p>While there is no premature appearance of melanophores in our mutants (A,B), by 5 dpf there are visibly more on the dorsal side of the head (C,D), and by 19 dpf, there is a marked increase in melanophore pigment cells both on the dorsal and lateral sides of the larvae (E,F). This increase in pigmentation is still seen at 30 dpf (G,H). Examination of transcript levels and expression patterns in 25 hpf embryos revealed an up-regulation of the early melanophore markers <i>tyrp1a</i> (I-K) and <i>zgc</i>:<i>91968</i> (L-N).</p

    <i>kctd15</i> mutants show up-regulation of several NC gene markers.

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    <p><i>foxd3</i> (A-C) and <i>sox10</i> (D-F) expression is indistinguishable between WT and mutants very early in NC development, but by 3-4s, expression of these markers shows both an expansion and up-regulation in expression, which persists at 8-9s. Other NC markers, including <i>dlx3b</i> (G), <i>sox9b</i> (H), <i>tfap2a</i> (I) and <i>snail1b</i> (J) also show up-regulation and expansion in our mutants at 8-9s. Additionally, an increase in NC cell number is apparent at 24hpf in <i>kctd15</i> mutants, as visualized in a <i>sox10-</i>GFP reporter construct (K).</p

    Kctd15 is required for proper jaw formation later in development.

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    <p>Alcian blue/Alizarin red staining does not reveal any early structural abnormalities in the patterning of <i>kctd15</i> mutant jaws (A,B). However, adult mutants exhibit shortening of several jaw elements, including the dentary, maxillary and frontal regions (C). While mRNA staining at 6 dpf for <i>col10a1a</i> showed no difference in patterning of early craniofacial bones (D,E), adult double mutants lack a properly formed maxilla bone (F,G; abnormalities are indicated by an asterisk and arrow).</p

    <i>kctd15</i> mutants show up-regulation of several NC gene markers.

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    <p><i>foxd3</i> (A-C) and <i>sox10</i> (D-F) expression is indistinguishable between WT and mutants very early in NC development, but by 3-4s, expression of these markers shows both an expansion and up-regulation in expression, which persists at 8-9s. Other NC markers, including <i>dlx3b</i> (G), <i>sox9b</i> (H), <i>tfap2a</i> (I) and <i>snail1b</i> (J) also show up-regulation and expansion in our mutants at 8-9s. Additionally, an increase in NC cell number is apparent at 24hpf in <i>kctd15</i> mutants, as visualized in a <i>sox10-</i>GFP reporter construct (K).</p
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