Phenotype of Knockdown netrin-1a Zebrafish Embryos Is Rescued by Inhibition of Apoptosis

(A–C) fli:egfp zebrafish embryos were injected at the one- to four-cell stage with control (ctl), splice (spl), or translation (tsl)-blocking netrin-1a, UNC5B, and DAP kinase morpholinos, alone or in combination. Phenotypes were analyzed from 24 to 54 hours postfertilization. Representative images of trunk vasculature are shown. Anterior is oriented toward the left. (A and B) The caspase inhibitor BAF restores parachordal vessel formation (asterisks) in (A) zebrafish embryos injected with spl-blocking netrin-1a morpholinos and intersegmental vessel formation in (B) zebrafish embryos injected with netrin-1a tsl morpholinos. (B) Similar phenotypical rescue was observed by simultaneous injection of UNC5B or DAP kinase and tsl-blocking netrin-1a morpholinos in zebrafish embryos. (A and B) Quantification is presented; p values are indicated (χ₂ test). (C) BAF treatment or injection of UNC5B-blocking morpholinos inhibit the increase in apoptosis observed after injection of tsl-blocking netrin-1a morpholinos in zebrafish embryos, as shown by TUNEL staining (left panels) or caspase-3 activity (right panel). Caspase-3 activity is expressed as a relative index referred to embryos injected with netrin-1a tsl morpholinos solely. Means and SEM are calculated from three independent experiments; p values are indicated (Student t′s test).

Effect of netrin-1a and UNC5B Morpholinos In Zebrafish Embryos
(A) Netrin-1b does not seem to be involved in developmental angiogenesis in zebrafish. Translation-blocking netrin-1b morpholino has no significant defect on developmental angiogenesis. Percentage of embryos without defects and total number of embryos analysed are indicated. Phylogenic study of netrin-1 otholog genes. ML on protein sequences, under JTT model, with 100 bootstrap replicates. Netrin-1 orthologs in zebrafish, netrin-1a and netrin-1b, are underlined.
(B) RT-PCR analysis of netrin-1a and UNC5B expression in zebrafish embryos.
(C) Third netrin-1a blocking morpholinos lead to similar vascular defects than both described in Figure 3. Percentage of embryos with ISVs defects and total number of embryos analysed are indicated.
(D) Netrin-1a silencing leads to increase caspase-3 activity in whole zebrafish embryos. Caspase-3 activity is expressed as a relative index referred to embryos injected with control MO. Means and s.e.m. are calculated from three independent experiments.