Neurofascin B Regulates Myelin Sheath Elongation and Stability(A and A′) Single time-point confocal images of a myelinating process in a Tg(sox10KalTA4, UAS mEGFP) control that makes contact with a target axon (A) and transitions into a recognizable myelin sheath (A′). Scale bar, 5 μm.(B) Average length of myelin sheaths immediately upon formation in control and nfascb MO-injected animals. Control median = 4.02, 25th percentile 3.45, 75th percentile 4.93, n = 91 from 13 animals and nfascb MO median = 3.67, 25th percentile 3.10, 75th percentile 4.63, n = 195 sheaths from 21 animals, p = 0.0611, Mann–Whitney test.(C and C′) Confocal images of a myelin sheath elongating over time during the period of sheath formation in a control animal. Arrowheads in all panels denote ends of myelin sheaths. Scale bar, 5 μm.(D) Average speed of individual sheath elongation in controls over a 12-h period, starting from the hour of their formation during the critical period. Each point represents a single sheath, imaged across 13 animals. No significant difference in speed of growth based on time of sheath formation, p = 0.8933, Kruskal-Wallis test.(E and E′) Single time-point confocal images of a myelin sheath elongating over time during the critical period of sheath formation in an nfascb MO-injected animal. Scale bar, 5 μm.(F) Average speed of sheath elongation in nfascb MO-injected animal over a 12-h period, starting from the hour of their formation during the critical period. Each point represents a single sheath, imaged across 21 animals. No significant difference in speed of growth based on time of sheath formation, p = 0.6301, Kruskal–Wallis test.(G and G′) Single time-point confocal images of a myelin sheath shrinking over time after the critical period in a nfascb MO-injected animal. Scale bar, 5 μm.(H) Speed of elongation of individual myelin sheaths in control and nfascb MO-injected animals for the first 6 h after their formation and the following 6 h. (control n = 34 sheaths in 8 animals, nfascb MO n = 66 sheaths in 13 animals: 0–6 h: control mean speed 0.77 ± 0.40 SD, nfascb MO mean 0.56 ± 0.41 SD, p = 0.0133, Mann–Whitney-test. 6–12 h: control mean speed 0.89 ± 0.42 SD, nfascb MO mean 0.185 ± 0.60 SD, p < 0.0001, Mann–Whitney test. Note the large number of sheaths shrinking in nfascb MO-injected animals between 6–12 h after sheath formation.(I and I′) Confocal images of a myelin sheath elongating in a wildtype animal. Scale bar, 10 μm.(J and J′) Confocal images of a myelin sheath elongating in an nfascbue56 mutant. Scale bar, 10 μm.(K) Speed of myelin sheath elongation in Tg(mbp:EGFP-CAAX) wildtype and nfascbue56 mutants (control mean speed 0.34 ± 0.40 SD, n = 118 from 6 animals, nfascbue56 mutant mean 0.06 ± 0.23 SD, n = 215 from 9 animals, p < 0.0001, Mann–Whitney test. 16.1% in controls and 36.4% in nfascbue56 represent shrinking myelin sheaths.(L) Cell body myelination in nfascbue56 mutants. Scale bar, 10 μm.

Mmp2 is localized between Z-discs in sarcomeres of embryonic and adult muscle. Confocal micrographs of skeletal muscle from 72 hpf embryos (A) or adults (B), and 500 nm thick cryosection of 72 hpf skeletal muscle (C) stained with anti-α-actinin (red) and anti-Mmp2 (green). Greyscale intensity profiles of both channels along a line drawn perpendicular to the sarcomeres are shown below each micrograph. Mmp2 immunoreactivity occurs at regularly spaced intervals precisely between the α-actinin-labeled Z-discs. Scale bars = 10 µm.

Nuclear distribution and shape indicate that hydrostatic pressure of the notochord is decreased in spzl mutants. ( A–B) Confocal images of cross sections of 14 dpf WT and spzl-/- larvae section. Vacuolated cells are labeled with SAG:gal4;UAS:GFP and nuclei are stained with DAPI. Scale bar = 50 µm ( C) Plot of vacuolated cell nuclear distribution for WT (red, n = 29) and spzl-/- (black, n = 32). ( D) Nuclear volume in WT and spzl-/- at 14 dpf. ( E–F) Reconstructions of nuclei in WT ( E) and spzl-/- ( F) and two different viewing angles. Scale bar = 20 µm ( G) Sphericity of WT and spzl-/- nuclei at 14 dpf. p-values were determined by an un-paired t-test using Welch’s correction.

Whole mount in situ and transverse section hybridization analysis of rab11ba in zebrafish embryos. A 1 k cell, lateral view. B 16 hpf, lateral view. B’ 16 hpf, dorsal view, arrowheads indicate neural tube. C 20 hpf, lateral view, arrowhead indicates brain. C’ 20 hpf, dorsal view, arrowheads indicate brain. D 24 hpf, lateral view, arrowhead indicate brain. E 36 hpf, lateral view, arrowhead indicates spinal cord. E’ 36 hpf, dorsal view, arrowhead indicates brain and head structure. F 48 hpf, lateral view, arrowhead indicates spinal cord. G 72 hpf, lateral view, arrowhead indicates brain. H 4 dpf, lateral view, red arrowhead indicates brain, green arrowhead indicates pectoral fins. H’ 4 dpf, asterisks indicate neuromasts. I 48 hpf, the trunk transverse section of the embryonic forebrain, arrowheads indicate cerebral cortex. I’ 48 hpf, the trunk transverse section of the embryonic midbrain, arrowheads indicate cerebral cortex. I” 48 hpf, the trunk transverse section of the embryonic hindbrain, arrowheads indicate cerebral cortex. J 48 hpf, lateral view, embryos stained with the sense rab11ba probe

Generation of capn3b null mutant allele. a Generation of the capn3bΔ19Δ14 mutant allele. Upper panel: diagram showing the genomic structure of the zebrafish capn3b gene and the two mutated sites in capn3bΔ19Δ14 generated by TALEN and CRISPR-Cas9 approaches, respectively. Vertical bar: exon; line connecting vertical bar: intron. Lower panel: highlighting the 19 bp deletion (red letters) in 1st exon generated by the TALEN approach (on the left) and 14 bps deletion (red letters) in 3rd exon by the CRISPR-Cas9 approach (on the right), respectively. The position of nucleotide in the capn3b open reading frame (ORF) from the translation start codon ATG is provided. C120, Capn3b activity center. b Predicted peptide encoded by the capn3bΔ19 (∆19) and capn3bΔ19Δ14 (Δ19Δ14) mutant mRNA, respectively. The Δ19 mutation leads to an early stop codon in the capn3b ORF and resulted in a 30 amino acids (aa) long polypeptide, however, the Δ19 mutation potentially allows the ATG codon encoding M94 residue of WT Capn3b to be used as an alternative translation start codon to translate an N-terminus truncated peptide which harbors the activity center C120. The Δ19Δ14 mutation creates a new early stop codon in the presumed variant initiated from M94 after C120, therefore, capn3bΔ19Δ14 is likely a null allele. c Western blotting analysis of Capn3b in WT, capn3b capn3bΔ19Δ14, capn3bΔ19 at 5dpf. β-Actin: loading control. d Immunostaining of Capn3b in WT and capn3bΔ19Δ14 mutant embryos at 5dpf. Scale bar: 50 μm

Sarcomeric Mmp2 begins to accumulate subsequent to the assembly of Z-disks. (A) Confocal section through the trunk musculature of a 24 hpf embryo posterior to the yolk extension, at the position at which myofibrils are differentiating. (B) Sarcomeric α-actinin (red) is beginning to become apparent as Z-bodies in the periphery of differentiating myocytes, but Mmp2 immunoreactivity (green) remains roughly homogeneously distributed. Greyscale intensity profiles of both channels along a line drawn through the periphery of a differentiating myocyte are shown below the micrograph. Scale bar = 10 µm.

Mmp2 is expressed ubiquitously from early development and accumulates in a striated pattern within the skeletal muscle. (A) Composite confocal projections of a 72 hpf embryo stained with anti-Mmp2 exhibiting labeling throughout the embryo with notable accumulation in the skeletal muscle (scale bar = 200 µm). (B) High magnification view of a single confocal section through the trunk musculature (indicated by the inset) showing strong labeling of the myotome boundary (upper left corner), and striated staining in myofibrils (scale bar = 10 µm). (C) RNASeq data showing absolute abundance of mmp2 transcripts in embryos from fertilization to five days post-fertilization (dpf). (D) Immunoblot of whole embryo homogenates (350 µg per lane) made from 2 hpf (cleavage), 5 hpf (50% epiboly), 12 hpf (early somitogenesis), 18 hpf (late somitogenesis), 24 hpf (prim-5), 48 hpf (long pec), and 72 hpf (protruding mouth) embryos probed with anti-Mmp2. Immunoreactivity is detected in embryos after 12 h of development, with bands at the expected mobility for full-length Mmp2 (72 kD) and stronger bands at 44 and 20 kD, which combine to give the expected size for activated Mmp2 (64 kD).

Similar spatial expression of znf143a and znf143b in 24hpf embryos. Antisense digoxigenin riboprobes against the znf143a and znf143b were used for whole-mount in situ hybridization assays. The probe for znf143b targeted the last exon of the coding region (approximately 186 nt corresponding to the last 61 amino acids) and the 3’UTR of the gene, while the probe for znf143a was designed solely against the 3’UTR. Embryos used were fixed and stained at 24hpf. The panels illustrate views from four different embryos for each probe, with the right-most panels showing an enlarged dorsal view of the head. We note that in all experiments, the znf143b probe produced fainter staining, most likely because of a smaller than optimal riboprobe that was necessary to ensure gene specificity. Specific head, brain and neural structures are identified with the following abbreviations: f, forebrain; m, midbrain; h, hindbrain; mhb, midbrain hindbrain boundary; c, cerebellum; scn, spinal cord neurons

etv2 expression analysis in wild‐type, etv2 ci32G + −/− and etv2 ci32Gt−/− embryos at the 20‐somite, A‐C, and 24 hpf stages, D‐F. Embryos were obtained from an incross of etv2 ci32Gt+/−; UAS:GFP +/+ parents and sorted based on their GFP fluorescence pattern. Antisense etv2 RNA probe which corresponds to the C‐terminal portion of the etv2 coding sequence and 3′UTR downstream of the Gal4 insertion side was used for in situ hybridization (see Experimental Procedures). A‐C, In non‐fluorescent wild‐type siblings (wt), strong etv2 expression is apparent in vascular progenitors in the anterior lateral plate mesoderm, presumptive progenitors of the anterior and common cardinal veins, and in vascular progenitors next to the tailbud (arrows). Weaker expression in the dorsal aorta (DA) is also apparent. Note the reduced etv2 expression in etv2 ci32Gt+/− embryos and nearly absent expression in etv2 ci32Gt−/− embryos. D‐F, In wild‐type siblings, strong etv2 expression is apparent in the cranial venous vasculature, including the primordial midbrain channel (PMBC) and the middle cerebral vein (MCEV), as well as the tail plexus region (arrow). Weaker expression in the DA is also apparent. Note the reduced etv2 expression in etv2 ci32Gt+/− embryos and nearly absent expression in etv2 ci32Gt−/− embryos