Soluble sialidase activity in zebrafish embryo and adult organs. Total extracts from wild-type embryos (A) and organs of 9-month-old adult fish (B) were assayed for soluble sialidase activity, as described in the Section 4 (n = 30). Extracts from various embryonic stages (12, 16, 24, 48, 72, 96 and 120 hpf) and from ovary, brain, heart and muscle were used as the enzyme sources. Assays were performed with up to 10 µg of total proteins. Sialidase activity was measured using 4-MU-NeuAc as a substrate at a pH of 5.6. Values are the mean ± SD of three independent experiments.

Effects of neu3.2 morpholino injection on zebrafish development. Representative images of the phenotype at 24 and 72 hpf obtained after the injection of neu3.2-MO. The injected embryos were compared with embryos injected with the standard morpholino (STD-MO). The embryos were injected with 1 pmol/embryo of neu3.2-splicing morpholino at 1–2 cell stages. For each injection experiment (n = 5), about 150 embryos were analyzed for morphology and classified with mild and severe phenotypes. The graphic in the bottom right indicates the percentage of embryos with mild and severe phenotypes. The results are expressed as mean ± SD of 5 independent experiments. (**** p < 0.0001 vs. injected embryos with standard morpholino-STD-MO—Unpaired; two-tailed t-test).

Rescue of the phenotype in neu3.2 morphants with Mus musculus Neu2 mRNA. Representative images, at 72 hpf, of the injected embryos with STD-MO (A) and with neu3.2 MO (1 pmol/embryo) alone (B) or together with 50 pg/embryo Neu2 mouse mRNA (C). The percentage of embryos with the different phenotypes (normal and severe) in the three categories of the analyzed embryos is shown in the graph (C). Neu 3.2 activity measured in the soluble fraction using 4-MU-NeuAc as a substrate at a pH = 5.6 in all three categories of analyzed embryos is shown in the graph (D). Three experiments were performed and more than 150 embryos of each type were analyzed. (**** p < 0.0001 vs. injected embryos with standard morpholino ST-MO—Unpaired; two-tailed t-test).

Downregulation of neu3.2 affects cardiac development. (A) Analysis of the cardiac marker cmlc2 by whole-mount in situ hybridization (WISH) in zebrafish embryos injected with STD-MO and injected with neu3.2-MO (1 pmol/embryo). Images are the anterior views of the whole mount with heart with dorsal up. Arrows point to the atrium (a) and ventricle (V), demonstrating their small size compared with the STD-MO-injected embryos. Ratios at the bottom left part of each picture specify the number of embryos showing the same staining pattern, compared to the total number of embryos used for each experiment. Two replicates were performed (n = 35). (B) Representative images of the cardiac area in the Tg(Bmp:EGFP) line STD-MO-injected and neu3.2-MO-injected (1 pmol/embryo at 48 hpf) embryos. Asterisk points out the reduction of fluorescence intensity and size of the heart in both conditions. Results are from one representative experiment with at least 25 embryos out of three independent replicates. (C) The graph shows the comparison of the heart rate (numbers of beats per minute) between STD-MO-injected and neu3.2-MO-injected (1 pmol/embryo) embryos and it is representative of one experiment (n = 25). The experiment was repeated three times. Results are expressed as the mean ± SD of three independent experiments. (**** p < 0.001 vs. the control group—embryos injected with standard morpholino-STD-MO).

neu3.2 knockdown causes defects in muscle formation. (A) Analysis of myod1 expression in neu3.2 morphants. Representative dorsal view images of the WISH analyses of myod1 expression of 14 and 22 hpf embryos injected with the standard morpholino (STD-MO) and neu3.2-MO. Results are from one representative experiment with embryos (n = 35) out of two independent replicates. Ratios at the bottom-left part of each picture specify the number of embryos showing the same staining pattern, compared to the total number of embryos used for each experiment. (B) The graph shows the percentages of hatched embryos at 72 hpf after injection with neu3.2 morpholino. Both categories were raised to 72 hpf and then analyzed for hatching. The X-axis shows the non-injected, injected with neu3.2-MO, with mild and severe phenotypes, and embryos rescued with murine Neu2 mRNA. The Y-axis reports the percentages of hatched embryos at 72 hpf. The results are expressed as the mean of 3 independent experiments, with 30 embryos for each experiment and each treatment. (* p and *** p < 0.001 vs. the control group—embryos injected with standard morpholino ST-MO). (C) Representative images with a light sheet microscope of Tg(Bmp:EGFP) embryos injected with the standard morpholino (STD-MO) and with neu3.2-MO (1.pmol/embryo) and rescued with murine Neu2 mRNA, observed at 48 hpf. A strong reduction in the fluorescence intensity is evident in neu3.2 morphants, whereas a recovery is detectable upon rescue with murine Neu2 mRNA. The results are from one representative experiment with at least 25 embryos out of three independent replicates. Magnification 10X.

Knockdown of neu3.2 interferes with myosin organization in slow myosin fibers and with myosepta. Light sheet imaging of the trunk longitudinal section after staining the actin fibers with phalloidin. Representative lateral views of the stained embryos showing a clear reduction in the total fluorescence intensity in neu3.2 morphants. Images were acquired with the same parameters as described the Section 4. Ratios at the bottom-left part of each picture specify the number of embryos showing the same staining pattern, compared to the total number of embryos used for each experiment. The experiment was repeated twice with n = 25.

Expression level analysis of different markers involved in muscle development in neu3.2 morphants and embryos injected with the standard morpholino (STD-MO). Gene expression was normalized using rpl13a as a reference gene expressed as the mRNA fold increase. Data are representative of three replicates and are shown as the mean of standard deviation (**** p < 0.001 vs. injected embryos with standard morpholino-STD-MO).

Locomotor analysis of neu3.2 morphants and murine Neu2-rescued embryos at different stages of development. Swimming performance of embryos at 48 hpf in the touch-evoked test. A significant progressive decrease in the percentage of neu3.2 morphants when compared to embryos injected with standard morpholino (STD-MO) or rescued–injected embryos was detected (**** p < 0.0001, Student’s t-test test). The results are representative of three independent experiments with 40 embryos per group. At 72 hpf, a significant percentage of neu3.2-MO-injected embryos exhibited a decreased number of tail flip movements when compared to STD-MO or rescued Neu2 mRNA-injected embryos (*** p < 0.001 vs. STD-MO; **** p < 0.0001 vs. STD-MO—Student’s t-test). At 6 dpf for a light–dark locomotion test, movements were reported as the mean ± SD of the total distance swam by the larvae (cm), calculated during both light and dark stimuli. The results are expressed as the mean ± SD of 3 independent experiments, with 12 surviving larvae for each experiment and for each treatment (**** p < 0.0001 vs. STD-MO; n.s.: not significative—Student’s t-test).