Analysis of Tg(ubi:switch) larvae shows fabp10a:CreERT2 leads to significant switching by 10 dpf even in the absence of tamoxifen treatment. (A,B) Switching in Tg(ubi:switch) zebrafish without (HepCreER−) and with (HepCreER+) the fabp10a:CreERT2 transgene, incubated with 4-hydroxytamoxifen (tamoxifen), ethanol, or egg water alone (no treatment) from 3 to 6 dpf and imaged at 6 dpf for EGFP and mCherry expression. Successful switching is indicated by loss of EGFP and gain of mCherry expression. (A) Representative images. Scale bars: 50 µm. Livers are outlined. (B) Scatter plot with bar graph quantifying switching in terms of ratio of hepatocytes that switched (mCherry+) relative to the total number of hepatocytes, ±s.d. P-values derived using Kruskal–Wallis non-parametric ANOVA followed by Dunn's multiple comparisons test. Graph shows combined data from two experiments. (C,D) Quantification of switching at 10 dpf in Tg(fabp10a:CreERT2);Tg(ubi:switch) (HepCreER+) and Tg(ubi:switch) (HepCreER−) larvae, incubated with tamoxifen, ethanol, or no treatment from 3 to 6 dpf. (C) Scatter plot with bar graph showing percentage of hepatocytes that switched (mCherry+) relative to the total number of hepatocytes, ±s.d. P-values obtained using Kruskal–Wallis non-parametric ANOVA followed by Dunn's multiple comparisons test. Graph represents combined data values from three experiments. (D) Representative images of HepCreER+ larvae. In TAM-treated zebrafish (top panels), most hepatocytes show switching (loss of EGFP and gain of mCherry expression); arrows indicate hepatocytes without switching. HepCreER+ zebrafish not treated with TAM (middle and bottom panels) show occasional cells with switching (arrowheads). Livers are outlined. Scale bars: 50 µm. (E) Quantification of switching at 20 dpf in HepCreER+ and HepCreER− larvae, incubated with tamoxifen, ethanol, or no treatment from 3 to 6 dpf. Scatter plot with bar graph shows percentage of hepatocytes that switched (mCherry+) relative to the total number of hepatocytes, ±s.d. P-values obtained using Sidak's multiple comparisons test following ordinary one-way ANOVA. This experiment was performed once. (F,G) Quantification of switching in adult (3 mpf) HepCreER+ and HepCreER− zebrafish, incubated with tamoxifen, ethanol, or no treatment from 3 to 6 dpf. (F) Scatter plot with bar graph shows the percentage of hepatocytes that switched (mCherry+) relative to the total number of hepatocytes, ±s.d. Graph represents combined data values from two experiments. P-values obtained using non-parametric Kruskal–Wallis test followed by Dunn's multiple comparisons test. (G) Representative images of liver (top and middle panels) or gut (bottom panel) cryosections showing complete switching (top panel) or no switching (middle and bottom panels). Scale bars: 40 µm.

Notochord vacuole morphology and arrangement is altered with increasing or decreasing mineralization. ( A, C) Live confocal images of fish treated with 0.0002% DMSO for 1 week at 6.0 mm and 6.5–7 mm respectively. ( B, D) Live confocal images of fish treated with 100 nM RA for 1 week at 6.0 mm and 6.5–7 mm respectively. Fish expressed nfatc:gal4;UAS:mcherry in the notochord and were stained with calcein to label centra. Scale bar = 100 µm. n = 3 for DMSO control and RA treatment. Notochord is outlined with a dotted white line. White arrows point to areas of increased mineralization, blue arrows point to the floor plate. Arrowheads indicate areas of increased vacuole fragmentation. Asterisks mark vacuolated cell stacking. ( E) Live confocal image of a 7.5 mm WT zebrafish stained with Cell Trace to visualize internal membranes and live alizarin to label mineralized bone, n = 3. ( F) Live confocal image of a 7.5 mm nobhu3718 stained with Cell Trace and alizarin red to label bone. n = 3. Black arrows point to vacuolated cells undergoing shape change. Asterisks mark stacked vacuolated cells. White arrows point to centra stained with alizarin red. v indicates rounded vacuoles that have not undergone shape change, fragmentation, or stacking. The notochord is outlined with a dotted fuchsia line. Scale bar = 100 µm. ( G–H) Cryosections of a 9.5 mm WT ( G) and nobhu3718 ( H) larvae stained with WGA (green) and alizarin red (magenta). Brackets indicate IVD domains. Arrows point to mineralized centra. The notochord is outlined with a dotted white line. Arrowheads point to mesenchymal condensations. Scale bar = 100 µm.

Up-regulation of the MARCKS family members and its interaction protein Hsp70.3 over-compensates the genetic loss of marcksb.(A, B) RT-qPCR analysis on the expression of marcksa, marcksb, marcksl1a, marcksl1b and hsp70.3 in the embryos indicated in the Fig at 1-cell stage (A) and shield stage (B). The data were presented as scatter plots with bar representing median value relative to respective transcript levels measured in wildtype embryos. “*”: P < 0.01, from Student’s t-test. (C) Overexpression of marcksa, marcksl1a, marcksl1b or hsp70.3 partially rescued the dorsalization defects in marcksb morphants. “n” represents the number of embryos we observed. (D) Co-immunoprecipitation revealed that Hsp70.3 had high binding affinity with Marcksb and moderate binding affinity with Marcksl11a and Marcksl1b, but relative low binding affinity with Marcksa. TCL: total cell lysis. The molecular mass of Marcks-myc is around 70KD. (E) The expression of szl was remarkably reduced in MZmarcksb injected with either hsp70_MO or marcksa_l1a_l1b_MO. The embryos are at shield stage and lateral view with dorsal to the right. (F) The percentage of embryos with normal-like and severely decreased expression of szl. “n” represents the number of embryos we observed. (G) Hsp70 interacts with Marcksa, Marcksl1a and Marcksl1b to maintain sufficient level of extracellular Bmp2b in MZmarcksb. (a-c) Compared with MZmarcksb embryos (a), knockdown of either hsp70 (b) or a combination of marcksa, marcksl1a and marcksl1b (c) remarkably reduced the level of extracellular Bmp2b in MZmarcksb. (H) Quantitative measurement of secreted Bmp2b in embryos of MZmarcksb, MZmarcksb injected with either hsp70_MO or marcksa_l1a_l1b_MO. The data were presented as scatter plots with median; “*”: P < 0.01, from Student’s t-test.

Caspr Regulates Myelin Sheath Elongation and Stability, but Not Targeting(A and B) Confocal images of single oligodendrocytes labeled mosaically using mbp:mCherry-CAAX in wildtype (A) and casprsa12772 mutants at 5 dpf. Scale bar, 10 μm.(C–E) Number of myelinated cell bodies (C), number of myelin sheaths (D), and the length of myelin sheaths (D), made by individual oligodendrocytes in wildtype, het, and casprsa12772 mutants.(C) Myelination of cell bodies is increased slightly in casprsa12772 mutant oligodendrocytes (wildtype median 0.0, 25th percentile 0.0, 75th percentile 0.0, n = 15 animals; casprsa12772/+ median 0.0, 25th percentile 0.0, 75th percentile 0.0, n = 35, casprsa12772 median 0.25, 25th percentile 0.0, 75th percentile 1.5 , n = 25 animals; wildtype versus casprsa12772/+ p = 0.8056, wildtype versus casprsa12772 p = 0.0305, casprsa12772/+ versus casprsa12772 p = 0.0156, all Mann–Whitney test.(D) Number of myelin sheaths per oligodendrocytes is similar in wildtype, casprsa12772/+ and casprsa12772 (wildtype mean 11.78 ± 5.14 SD, n = 15 animals, casprsa12772/+ mean 11.19 ± 3.26 SD, n = 33 animals, casprsa12772 mean 12.00 ± 3.54 SD, n = 24; ANOVA = 0.712).(E) Myelin sheath length is reduced in casprsa12772 mutants (wildtype mean 29.14 ± 1.71 SEM, n = 15, casprsa12772/+ mean 27.58 ± 1.31 SEM, n = 33 animals, casprsa12772 mean 23.55 ± 1.30 SEM, n = 24 animals; wildtype versus casprsa12772/+ p = 0.4954, wildtype versus casprsa12772 p = 0.0124, casprsa12772/+ versus casprsa12772 p = 0.0374, all t test).(F and G) Confocal images of the dorsal horn of the spinal cord in wildtype (F) and Caspr mutant (G) mice, indicating the region indicated by dashed lines wherein myelinated cell bodies were searched for. Scale bar, 50 μm.(F′ and G′) Higher magnification views of regions within the dorsal horn of wild type (F′) and Caspr mutant (G′) animals stained with antibodies that recognize myelin basic protein (green), NeuN (red), and DAPI (blue). Scale bar, 5 μm.(H) Graph showing that essentially no myelinated cell bodies were observed in wild type or Caspr mutant animals. Wild type mean 0.2800 ± 0.049 SEM, n = 5 (10 sections per mouse), Caspr−⁄− 0.257 ± 0.083 SEM, n = 5 (10 sections per mouse), p = 0.8147, t test.(I and J) Confocal images of teased fiber preparations taken from wild type (I) and Caspr mutant (J) mice, stained with antibodies that detect Kv1.1 (red), Neurofascin 186 (green), and neurofilament 200 (red). Scale bars, 25 μm.(K) Quantitation of myelin sheath length in Caspr mutant mice. Caspr internode lengths are reduced compared to wild type; wild type mean 310.7 ± 5.32 SEM, n = 5, Caspr 226.5 ± 8.96 SEM, n = 5 animals, 50 internodes per mouse, p < 0.0001, t test.(L and L″) Confocal images of a myelin sheath elongating in a wildtype Tg(mbp:EGFP-CAAX) animal. Scale bar, 10 μm.(M and M′) Confocal images of a myelin sheath elongating in a casprsa12772 mutant. Scale bar, 10 μm.(N) Speed of myelin sheath elongation in wildtype and casprsa12772 mutants: wildtype mean 0.36 ± 0.35 SD, n = 122 sheaths from 6 animals, casprsa12772 mean 0.16 ± 0.31 SD, n = 151 sheaths from 7 animals, p < 0.0001, Mann–Whitney test. 14.8% in controls and 28.5% in nfascbue56 represent shrinking myelin sheaths.

Plvapb limits the transfer rate of blood-borne proteins into the hypophysis parenchyma. (A) Confocal maximal intensity images showing the hypophyseal capillary loop in plvapb+/+ and plvapb−/− double transgenic Tg(l-fabp:DBP-EGFP;kdrl:mCherry-caax) zebrafish larvae (5 dpf). Scale bars: 10 µm. (B,C) Morphological analysis of hypophyseal capillary loop parameters, including roundness index [width (x)/length (y)] (B) and area (C). No significant differences were found between plvapb+/+ (n=18) and plvapb−/− (n=23) fish (ns, not significant; Student's t-test). (D) Quantification of accumulated fluorescence intensity of the extravasated DBP-EGFP inside the hypophyseal capillary loop (dashed line in A, left panel) of fixed plvapb+/+ (n=16) and plvapb−/− (n=23) larvae. No significant difference was found between the fish (ns, not significant; Student's t-test). (E) Schematic representation of the fluorescence recovery after photobleaching (FRAP) of extravasated DBP-EGFP in the hypophysis of live zebrafish larval. ROI, region of interest. (F) Real-time FRAP measurements of the intensity of extravasated DBP-EGFP signal in live plvapb+/+ (n=8) and plvapb−/− (n=10) transgenic Tg(l-fabp:DBP-EGFP;kdrl:mCherry-caax) larvae (4 dpf). (G) T1/2 of the fluorescence recovery after bleaching of DBP-EGFP. T1/2 value (calculated using ImageJ FRAP Profiler tool) is significantly lower in the plvapb−/− mutant, indicating higher extravasation rate of blood-borne protein in mutant versus wild-type fish (*P<0.05; Student's t-test). a.u., arbitrary units. Data are mean±s.e.m.

MZmarcksb showed genetic compensation on BMP signaling.(A, B) Injection of marcksb_MO in MZmarcksb did not cause defect of dorsalization. (A) morphological observation of MZmarcksb and marcksb_MO injected MZmarcksb. Up panels, field view of embryos at 12 hpf; lower panels, representative embryos at 30 hpf. (B) WISH analysis showed that the transcriptional levels of downstream targets of BMP signaling—szl and ved were at comparable level between MZmarcksb and marcksb_MO injected MZmarcksb. (C) The overall intensity of p-Smad1/5/9 at ventral region in MZmarcksb was slightly increased when compared with wildtype. (D) The normalized fluorescent intensity of P-Smad1/5/9. Error bars of light blue and light red show S.E.M. (E) The extracellular mCherry-Bmp2b was slightly increased in the MZmarcksb compared with wildtype embryos. (F) Quantitative measurement of secreted Bmp2b in wildtype and MZmarcksb embryos. The data were presented as scatter plots with median; *”: P < 0.01, from Student’s t-test. (G) Although knockdown of chd in wildtype embryos only resulted in moderate ventralization (V1-V2), knockdown of chd in MZmarcksb embryos resulted in severe ventralization (V3-V4). V1-V4, ventralization type 1 to type 4 embryos; the statistical data are shown in the bar graphs with the number of observed embryos indicated right. (H) After injection of chd_MO, the up-regulation of BMP signaling activity was much more robust in MZmarcksb than that in wildtype embryos shown by WISH analysis of szl (a vs b) and ved (c vs d). The embryos are at shield stage and animal-pole view with dorsal to the right; (I) The percentage of embryos with normal-like, increased, and dramatically-increased expression of szl and ved. “n” represents the number of embryos we observed.

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

Divergent expression of csf1ra and csf1rb during zebrafish early development and in scales of the adult zebrafish. Whole-mount in situ hybridization of csf1ra (A) and csf1rb (B). (A) csf1ra is expressed in cells of the pigment and hematopoietic lineages at 2 dpf; inset shows the trunk in different focal plane with pigment cells precursors (arrowheads) and hematopoietic cells (arrows). (B) In contrast, csf1rb is expressed at only low levels in the heart (arrow) at 2 dpf. (C) Expression of Tg[cstk:DsRed] in osteoclasts localizes at areas of remodeling, as shown by increased calcein staining (blue) at the periphery of the remodeled pit. (D,E) Analysis of expression of csf1r paralogues in isolated cells and from adult skeletal tissues. (D) Expression of csfr1a and csf1rb in isolated adult osteoblasts (sp7+ cells) and osteoclasts (ctsk+ cells) from wild-type zebrafish. (E) Detection of both paralogues in isolated tissues [fin, teeth and vertebra (vert)].

The expression of polster genes in the klf17-deficient embryos at the bud stage. The expression of polster markers he1.1 (a,b), ctsl1b (c,d), cd63 (e,f) and klf17 (g,h) was examined by WISH at the bud stage. (a,c,e,g) Wild-type embryos (klf17+/uy23). (b,d,f,h) klf17-deficient embryos (klf17uy23/uy23). Lateral view, anterior left. Arrowheads indicate the position of the polster. After taking pictures, genotyping of individual embryos was performed by genomic PCR. Scale bar, 200 μm.