Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: tbx6l and tbx16 are redundantly required for posterior paraxial mesoderm formation during zebrafish embryogenesis

doi: 10.1002/dvdy.24547

Figure Lengend Snippet: (A–F) In situ hybridization for isl1 (A–C) and sox2 (D–F) at 24 hpf. (G–L) Confocal projections of embryos labeled for acetylated tubulin (green), fast muscle (F310; red), and myosin heavy chain (A4.1025; blue) in lateral (G–I) and dorsal (J–L) views. Not shown are tbx6l single mutant embryos as they are indistinguishable from wild-type embryos. Scale bar in A (for A–F) and G (for G–I) is 100 µm, and in J (for J–L) is 50 µm.

Article Snippet: Embryos were rehydrated, blocked for 4 hours at room temperature, then incubated overnight at 4°C in primary antibody solution containing anti-acetylated tubulin [1:1000; Sigma, T6793], F310 [1:1000; DSHB], and A4.1025 [1:000; DSHB].

Techniques: In Situ Hybridization, Labeling, Mutagenesis

Journal: bioRxiv

Article Title: Functional assessment of a kcnb1 knock-out zebrafish to model KCNB1 -related neurodevelopmental and epileptic disorders

doi: 10.1101/2024.07.03.601913

Figure Lengend Snippet: A. Images showing that kcnb1 +/− and kcnb1 −/− embryos and larvae do not show gross morphological changes at 28 hours post-fertilization (hpf) and 6 dpf (scale bar: 300 µm). B. Quantification of major morphological aspects at 48 hpf and 6 dpf including measurement of body length and head surface. The kcnb1 mutant zebrafish models ( kcnb1 +/− and kcnb1 −/− ) do not show any significant change in each parameter at different developmental stages as compared to kcnb1 +/+ (see ; body length at 6 dpf: kcnb1 +/+ = 3.45 ± 0.03 mm; kcnb1 +/− = 3.82 ± 0.02 mm; kcnb1 −/− = 3.62 ± 0.04 mm; +/+ vs mutants: p>0.5; N = 4 repeats; n = 28-38 ZF/genotype; one-Way ANOVA with Bonferroni post-hoc test; ns: non-significant). Values represent the mean ± standard error of the mean (SEM). C. Transversal slices of 48 hpf and 4 dpf larvae expressing cells (DAPI, blue) labelled with anti-HuC (red), a pan-neuronal marker, showing no major difference of anatomical brain regions between mutants and wild-type fish (scale bar: 50 µm; magnification 20x). D and E. Whole-mount images of embryonic (48 hpf) and larvae (6 dpf) zebrafish immunostained with (D) anti-3A10 marker to identify Mauthner cells (see ; dorsal view; 3D reconstruction; scale bar: 50 µm; magnification 20x) and (E) anti-acetylated tubulin marker to identify global circuits of neuronal fibers (lateral and dorsal view; 3D reconstruction; magnification 20x and scale bar at 50 μm; magnification 40x and scale bar at 20 µm). The partial or complete loss of kcnb1 does not affect the neuronal brain density at different early stages of development (n = 5-7 ZF/genotype/developmental stage).

Article Snippet: Primary antibodies used were mouse anti-acetylated tubulin antibody (1:100, Sigma-Aldrich, USA, #T7451), mouse anti-3A10 antibody (1:100, DSHB, Iowa City, Iowa, USA, #3A10), mouse anti-F310 antibody (1:100, DSHB, USA, #F310), mouse anti-F59 antibody (1:20, DSHB, USA, #F59), mouse anti-Znp1 antibody (1:100, DSHB, USA, #znp1) and mouse anti-c-Fos antibody (1:100, Santa Cruz Biotechnology, Dallas, Texas, USA, #sc-166940).

Techniques: Mutagenesis, Expressing, Marker

Journal: bioRxiv

Article Title: Functional assessment of a kcnb1 knock-out zebrafish to model KCNB1 -related neurodevelopmental and epileptic disorders

doi: 10.1101/2024.07.03.601913

Figure Lengend Snippet: A. Images showing that kcnb1 +/− and kcnb1 −/− embryos and larvae do not show gross morphological changes at 28 hours post-fertilization (hpf) and 6 dpf (scale bar: 300 µm). B. Quantification of major morphological aspects at 48 hpf and 6 dpf including measurement of body length and head surface. The kcnb1 mutant zebrafish models ( kcnb1 +/− and kcnb1 −/− ) do not show any significant change in each parameter at different developmental stages as compared to kcnb1 +/+ (see ; body length at 6 dpf: kcnb1 +/+ = 3.45 ± 0.03 mm; kcnb1 +/− = 3.82 ± 0.02 mm; kcnb1 −/− = 3.62 ± 0.04 mm; +/+ vs mutants: p>0.5; N = 4 repeats; n = 28-38 ZF/genotype; one-Way ANOVA with Bonferroni post-hoc test; ns: non-significant). Values represent the mean ± standard error of the mean (SEM). C. Transversal slices of 48 hpf and 4 dpf larvae expressing cells (DAPI, blue) labelled with anti-HuC (red), a pan-neuronal marker, showing no major difference of anatomical brain regions between mutants and wild-type fish (scale bar: 50 µm; magnification 20x). D and E. Whole-mount images of embryonic (48 hpf) and larvae (6 dpf) zebrafish immunostained with (D) anti-3A10 marker to identify Mauthner cells (see ; dorsal view; 3D reconstruction; scale bar: 50 µm; magnification 20x) and (E) anti-acetylated tubulin marker to identify global circuits of neuronal fibers (lateral and dorsal view; 3D reconstruction; magnification 20x and scale bar at 50 μm; magnification 40x and scale bar at 20 µm). The partial or complete loss of kcnb1 does not affect the neuronal brain density at different early stages of development (n = 5-7 ZF/genotype/developmental stage).

Article Snippet: Primary antibodies used were mouse anti-acetylated tubulin antibody (1:100, Sigma-Aldrich, USA, #T7451), mouse anti-3A10 antibody (1:100, DSHB, Iowa City, Iowa, USA, #3A10), mouse anti-F310 antibody (1:100, DSHB, USA, #F310), mouse anti-F59 antibody (1:20, DSHB, USA, #F59), mouse anti-Znp1 antibody (1:100, DSHB, USA, #znp1) and mouse anti-c-Fos antibody (1:100, Santa Cruz Biotechnology, Dallas, Texas, USA, #sc-166940).

Techniques: Mutagenesis, Expressing, Marker

Journal: bioRxiv

Article Title: Functional assessment of a kcnb1 knock-out zebrafish to model KCNB1 -related neurodevelopmental and epileptic disorders

doi: 10.1101/2024.07.03.601913

Figure Lengend Snippet: Fish were labelled with anti-3A10 and the length of Mauthner cell body (M-cell) was measured between the two extremities as represented with the arrow. The average value of the two M-cell bodies of each fish was used for quantification. Both kcnb1 knock-out zebrafish models ( kcnb1 +/− and kcnb1 −/− ) do not present difference in the length of M-cell body at 48 hpf as compared to the control condition ( kcnb1 +/+ ). kcnb1 +/+ = 39.58 ± 2.26 µm; kcnb1 +/− = 37.52 ± 0.86 µm; kcnb1 −/− = 43.55 ± 1.96 µm; +/+ vs mutants: p>0.5; n = 3-5 ZF/genotype; Mann-Whitney test; ns: non-significant. Values represent the mean ± standard error of the mean (SEM).

Article Snippet: Primary antibodies used were mouse anti-acetylated tubulin antibody (1:100, Sigma-Aldrich, USA, #T7451), mouse anti-3A10 antibody (1:100, DSHB, Iowa City, Iowa, USA, #3A10), mouse anti-F310 antibody (1:100, DSHB, USA, #F310), mouse anti-F59 antibody (1:20, DSHB, USA, #F59), mouse anti-Znp1 antibody (1:100, DSHB, USA, #znp1) and mouse anti-c-Fos antibody (1:100, Santa Cruz Biotechnology, Dallas, Texas, USA, #sc-166940).

Techniques: Knock-Out, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Functional assessment of a kcnb1 knock-out zebrafish to model KCNB1 -related neurodevelopmental and epileptic disorders

doi: 10.1101/2024.07.03.601913

Figure Lengend Snippet: A. Whole-mount images of 48 hpf-zebrafish obtained with a brightfield microscopy and treated with Phalloidin preventing actin depolymerization to identify general muscle fiber organization ( and 8A b ). Fish were then labelled with anti-F310 and anti-F59 markers to differentiate, respectively, fast and slow muscle fibers ( and 8A d ). Control fish ( kcnb1 +/+ ) and kcnb1 mutant models ( kcnb1 +/− and kcnb1 −/− ) display well organized muscle structures with typical parallel line pattern at 48 hpf (n = 4-8 ZF/genotype; 3D reconstruction; lateral view; magnification 20x; scale bar: 50 μm). B. Quantification of the anisotropy of fibrillar structures in raw images of Phalloidin, F310 and F59 labelling using the plugin FibrilTool from the software ImageJ . A Gaussian filter was applied on images and the anisotropy was analyzed reflecting the organization of fibers following the convention: 0 for no order and 1 for perfectly ordered (parallel fibrils) (anisotropy of fast muscles (F310): kcnb1 +/+ = 0.443 ± 0.02; kcnb1 +/− = 0.387 ± 0.06; kcnb1 −/− = 0.51 ± 0.02; +/+ vs mutants: p>0.5; n = 5-12 ZF/genotype/marker; Mann-Whitney test; ns: non-significant). Values represent the mean ± standard error of the mean (SEM). C. Plot profile illustrating the relative distance between fibers for each muscle fibers labelling (Phalloidin, F310 and F59). The results show similar profiles between control and both mutant conditions reflecting well organized muscle fibers (n = 4-8 ZF/genotype/marker). D. Primary motoneuron axons of controls and kcnb1 mutant zebrafish at 48 hpf were immunostained with anti-Znp1 marker (green) and acetylcholine receptors (AchR) were labelled with Bungarotoxin (BTX, red) (n = 4-8 ZF/genotype, magnification 20x, scale bar: 50 μm). At 48 hpf, to ef1α mRNA expression and kcnb1 +/+ fish are considered as the reference value (relative mutant fish ( kcnb1 +/− and kcnb1 −/− ) present similar motor axons length and branching as compared to WT fish associated with colocalization with AchR puncta identified along axons, quantified with Imaris software and represented by yellow spots, indicating functional synapses. E. Quantification of motor axon length of fish at 48 hpf. An average of eight motor axon length per fish was measured and a ratio was applied according to the spinal cord thickness. The total length quantification did not show any difference in motor axon size between mutant zebrafish models and the control condition ( kcnb1 +/+ = 2.38 ± 0.11; kcnb1 +/− = 2.43 ± 0.06; kcnb1 −/− = 2.55 ± 0.14; +/+ vs mutants: p>0.5; n = 7–9 ZF/condition; Mann-Whitney test; ns: non-significant). Values represent the mean ± standard error of the mean (SEM). F and G. Total RNAs were extracted from fish at 6 dpf and RT-PCR was performed to determine the transcript level of (F) acetylcholinesterase (AchE) and of (G) subunits α1, γ, ε (chrn) of AchR. Data are normalized to ef1α mRNA expression and the condition kcnb1 +/+ is considered as the reference value (relative fold change = 1). Both mutant conditions ( kcnb1 +/− and kcnb1 −/− ) presented significant decrease of AchE expression, reflecting a neurotransmission recycling defect at the NMJ level . However, the composition of AchR subunits is similar between conditions . N = 3; n = 30/sample; Mann- Whitney test; ns: non-significant; *p<0,05; **p<0,01. Values represent the mean ± standard error of the mean (SEM). chrn: cholinergic receptor nicotinic.

Article Snippet: Primary antibodies used were mouse anti-acetylated tubulin antibody (1:100, Sigma-Aldrich, USA, #T7451), mouse anti-3A10 antibody (1:100, DSHB, Iowa City, Iowa, USA, #3A10), mouse anti-F310 antibody (1:100, DSHB, USA, #F310), mouse anti-F59 antibody (1:20, DSHB, USA, #F59), mouse anti-Znp1 antibody (1:100, DSHB, USA, #znp1) and mouse anti-c-Fos antibody (1:100, Santa Cruz Biotechnology, Dallas, Texas, USA, #sc-166940).

Techniques: Microscopy, Control, Mutagenesis, Software, Muscles, Marker, MANN-WHITNEY, Expressing, Functional Assay, Reverse Transcription Polymerase Chain Reaction

Journal: The European Journal of Neuroscience

Article Title: Embryonic motor activity and implications for regulating motoneuron axonal pathfinding in zebrafish

doi: 10.1111/j.1460-9568.2008.06418.x

Figure Lengend Snippet: Information pertaining to primary antibodies

Article Snippet: The monoclonal antibodies F59 (1 : 50 dilution; ; Developmental Studies Hybridoma Bank, University of Iowa), F310 (1 : 250; ; Developmental Studies Hybridoma Bank, University of Iowa) and 4D9 (1 : 200 dilution; Patel et al. , 1989 ; Developmental Studies Hybridoma Bank, University of Iowa) were used to label slow muscle fibers (F59), fast muscle fibers (F310) and to detect engrailed (4D9), a marker of muscle pioneers in zebrafish.

Techniques: Concentration Assay, Marker

Journal: The European Journal of Neuroscience

Article Title: Embryonic motor activity and implications for regulating motoneuron axonal pathfinding in zebrafish

doi: 10.1111/j.1460-9568.2008.06418.x

Figure Lengend Snippet: 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.

Article Snippet: The monoclonal antibodies F59 (1 : 50 dilution; ; Developmental Studies Hybridoma Bank, University of Iowa), F310 (1 : 250; ; Developmental Studies Hybridoma Bank, University of Iowa) and 4D9 (1 : 200 dilution; Patel et al. , 1989 ; Developmental Studies Hybridoma Bank, University of Iowa) were used to label slow muscle fibers (F59), fast muscle fibers (F310) and to detect engrailed (4D9), a marker of muscle pioneers in zebrafish.

Techniques: Immunohistochemistry, Labeling, Expressing