Loss of drl and lrrc15 leads to the defect of hematopoietic and/or vascular development. (A) Relative expression levels of drl and lrrc15 in isolated fli1a:GFP (+) cells from WT or jam3bsa37 embryos. Error bars represent s.d. (n=3 each). (B) Expression of drl in the PLPM of WT or jam3bsa37 embryos. (C,D) Expression of lrrc15 in WT or jam3bsa37 embryos. Black arrowheads indicate the vascular cord (C) or DA (D). White arrows indicate primitive erythrocytes in the PBI (D, left). A dotted circle outlines the DA, and a black arrow indicates an lrrc15-expressing cell in the ventral floor of the DA (D, right). (E) Expression of runx1, fli1a, gata1a and npas4l in embryos uninjected or injected with lrrc15 MO or drl MO. Black arrowheads in E indicate the expression domain of each gene. Numbers in bottom right of panels indicate the number of embryos showing the displayed expression pattern over the total number of analyzed embryos. lm, lumen. Scale bars: 5 μm (D, right panels); 100 μm (B,C,E; D, left panels).

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

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

Defective development of capn3b mutant zebrafish under environment stress. a, b WISH using the fabp10a probe on 3dpf- and 5dpf-old WT and capn3bΔ19Δ14 embryos grown at the low (60 embryo/dish) (left panel) and high (120cm2/dish) (right panel) rearing density in a 9 cm-diameter dish (a). In (b), the data of liver sizes were compared between 3dpf- and 5dpf-old WT or between 3dpf- and 5dpf-old capn3bΔ19Δ14 mutant embryos under low (L) and high (H) rearing intensity. c WISH using the fabp10a and trypsin probes on WT and capn3bΔ19Δ14 embryos at 2dpf and 3dpf. Embryos were shifted to 34.5 °C at 12 hpf till the time of sample harvesting. Numerator/denominator: number of embryos displayed the shown phenotype over total number of genotyped embryos. d Photo images showing the curved body phenotype displayed by the 3dpf-old capn3bΔ19Δ14 mutant but not WT embryos grown at 34.5 °C. e Body lengths of 6-, 8- and 12-months-old WT and capn3bΔ19Δ14 fish. f LBR of 6- and 12-months-old WT and capn3bΔ19Δ14 fish. Student’s T-test for statistical analyses, *, p < 0.05; ***, p < 0.001; ****, p < 0.0001

Maternal effect of larp6a−/− and larp6a−/−;larp6b−/− on oogenesis. (A) Bright-field images of 5 dpf larvae from the indicated crosses. Fish are shown with anterior towards the left and dorsal upwards. Inflated swim bladders show that larvae have swimming and swallowing capacity. (B) Slow myosin immunodetection in 48 hpf larvae from a larp6a−/−;larp6b−/− female crossed to a larp6a+/−;larp6b+/− male reveal no differences in slow muscle fibre formation. Images are centred on somite 17/18 and the graph shows the number of slow fibres per myotome±s.d., tested using Welch's ANOVA on SPSS. (C) Bright-field images of lays at 3 hpf from wild-type, larp6a−/−, larp6b−/− and larp6a−/−;larp6b−/− females (upper panels) or males (lower panels) crossed to wild-type AB. Embryos from males of any genotype or wild-type females show a fully elevated chorion (ch), translucent and smooth yolk (y), tall and symmetrical blastodisc (bd), large chorion diameter (brackets), and large subchorionic space (asterisk). All embryos from maternal larp6a−/− mutants have reduced chorion diameter, reduced subchorionic space (black lines) surrounding a yolk cell of unaltered size (red lines) but more opaque and uneven. Lays from larp6b−/− females are indistinguishable from wild type. Lays from larp6a−/−;larp6b−/− females are like larp6a−/− embryos, but have even smaller chorion diameter (brackets). (D) Chorion diameter cumulative frequency distribution curves. Light colours indicate individual clutches strong colour indicates the mean for each genotype. (E) Average of the mean chorion diameters of n clutches from separate females of indicated maternal genotype±s.d. One-way ANOVA with Tukey's post-hoc test on SPSS. Scale bars: 1 mm in A,C; 100 µm in B.

Jam3b regulates the integrin signaling pathway to form HSCs. (A) Fluorescent image of a fli1a:Gal4; UAS:mCherry-itgb1aDN embryo. (B) Expression of runx1 in the DA of mCherry-itgb1aDN (−), or mCherry-itgb1aDN (+) embryos uninjected or injected with ca-faka mRNA. (C) Expression of lrrc15 in the vascular cord of mCherry-itgb1aDN (−) or mCherry-itgb1aDN (+) embryos. (D) Expression of runx1 in the DA of mCherry-itgb1aDN (+) embryos uninjected or injected with lrrc15 mRNA. (E) Time-lapse images of fli1a:GFP in WT or jam3bsa37 embryos. Dotted lines indicate the boundary of eleventh and twelfth somite at each time point. White arrowheads indicate a subset of fli1a:GFP (+) cells that show a rounded morphology. Data are representative of four embryos for each genotype. (F) Expression of lrrc15 in the vascular cord of WT, or jam3bsa37 embryos uninjected or injected with drl mRNA, ca-faka mRNA, or both drl and ca-faka mRNA. (G) Expression of runx1 in the DA of WT, or jam3bsa37 embryos uninjected or injected with ca-faka mRNA or both drl and ca-faka mRNA. (H) Expression of lrrc15 in the vascular cord of WT embryos treated with DMSO or wortmannin. (I) Expression of runx1 in the DA of WT embryos treated with wortmannin, either uninjected or injected with lrrc15 mRNA. Percent distribution of runx1 expression in I is shown in Fig. S8C. Black arrowheads indicate the expression domain of each gene. Numbers in bottom right of panels indicate the number of embryos showing the displayed expression pattern over the total number of analyzed embryos. Scale bars: 50 μm (E); 100 μm (A-D,F-H).

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.

Zebrafish disease modeling. a Schematic drawing of the ugp2a and ugp2b loci in zebrafish and the generated mutations. b Confocal images (maximum projection of confocal Z-stacks) of the brain of wild type (left) and ugp2aΔ/Δ; ugp2bΔ/Δ mutant zebrafish larvae (right), both in an slc1a2b-citrine reporter background, at 4 days post-fertilization (dpf). The lower panels are higher magnifications of the boxed regions indicated in the upper panels. Scale bar in upper panel is 100 µm, in lower panel 20 µm. In upper panel, Z = 45 with step size 4 µm; in lower panel, Z = 30 with step size 2 µm. c Enzymatic activity in ugp2 double mutant zebrafish larvae at 4 and 5 dpf, compared to wild-type age-matched controls, showing reduced Ugp2 enzyme activity in double mutant zebrafish. d qRT-PCR for the neuronal activity marker c-fos in wild type and ugp2 double mutant larvae at 3 dpf. For each group, 2 batches of 12 larvae were pooled. Shown is the mean fold change for the indicated genes compared to wild type, normalized for the housekeeping gene gapdh. Error bars represent SEM; ***p < 0.001, unpaired t test, two tailed. e Representative graph of a locomotion assay showing the total distance moved by larvae during the dusk–dawn routine (total time: 3 h 12 min), n = 24 larvae per genotype. Gray shading shows the standard error of the mean. f Quantification of the total distance moved throughout the experiment from e excluding the dark period. g Quantification of the number of observed spontaneous eye movements during a 2-min observation in wild type and ugp2 double mutant larvae at 4 dpf. Each dot represents one larva; shown is the average and SD; ***p < 0.001, t test, two tailed. h Quantification of the frequency of movements at a speed of > 15 mm/s, for wild-type control and ugp2 double mutant zebrafish larvae at 4 dpf, treated with mock control or with 0.04 nM or 0.4 nM 4-AP during a 35-min observation. Each dot represents a single larva; results of two experiments are shown, within total 24 larvae per condition. i Quantification of the movement duration at a speed of > 15 mm/s, for wild-type control and ugp2 double mutant zebrafish larvae at 4 dpf, treated with mock control or with 0.04 nM or 0.4 nM 4-AP during a 35-min observation. Each dot represents a single larva; results of two experiments are shown, with in total 24 larvae per condition. *p < 0.05, two-way ANOVA with Bonferoni post test

Whole mount in situ and transverse section hybridization analysis of rab11bb in zebrafish embryos. A 64 cell, lateral view. B 20 hpf, lateral view, arrowheads indicate brain and spinal cord. C 24 hpf, lateral view, arrowheads indicate brain and spinal cord. D 36 hpf, lateral view, arrowheads indicate spinal cord. D’ 36 hpf, dorsal view, red arrowheads indicate spinal cord, green arrowheads indicate pronephric duct. E 48 hpf, lateral view, arrowhead indicates brain. E’ 48 hpf, dorsal view. F 72 hpf, lateral view, arrowhead indicates ear. F’ 72 hpf, dorsal view. G 4 dpf, lateral view, arrowhead indicates pharyngeal arches. G’ 96 hpf, dorsal view, arrowhead indicates retina. H 48 hpf, the trunk transverse section of the embryonic forebrain, arrowheads indicate cerebral cortex. H’ 48 hpf, the trunk transverse section of the embryonic midbrain, arrowheads indicate cerebral cortex. H” 48 hpf, the trunk transverse section of the embryonic hindbrain, arrowheads indicate cerebral cortex. I 48 hpf, lateral view, embryos stained with the sense rab11bb probe