|ZFIN ID: ZDB-IMAGE-140902-108|
Toxicity of rtn4a morpholino-mediated knockdown. (A) Impaired development and viability after injection of rtn4-morpholino (MO). Embryos were microinjected at cell stages 1 to 4 and analyzed at 6 hpf and 15 hpf. Higher doses of rtn4a-MO resulted in delayed gastrulation at 6 hpf and reduced viability at 15 hpf compared to control embryos (arrowhead). (B)rtn4a and rtn4b MOs were microinjected at 2.5 or 5.0 ng. Mortality, development and viability were evaluated for all experimental groups in at least three experiments. Embryos showing a curly tail phenotype upon Rtn4a knockdown were excluded from the experimental group. (C) Quantification of embryos with reduced anterior brain structures was carried out after knockdown of each Rtn4a isoform. Control (n = 95), rtn4a-l MO1 (n = 78), rtn4a-m MO1 (n = 23), rtn4a-n MO1 (n = 33) and rtn4a (l, m and n) MO1 (n = 78). (D) A second rtn4b-MO sequence (rtn4b-MO2) elicited the same phenotype as the rtn4b-MO1 sequence. At 15 hpf, the forebrain was flattened and the sizes of the head and eye anlage were reduced. At 1 dpf, rtn4b-MO2-injected embryos were smaller than control embryos, with reduced heads and eyes. Embryos also had an abnormally curved notochord. At 2 dpf, rtn4b morphants had shortened fore- and midbrain regions. At 3 dpf, rtn4b morphants developed a thinner tail, which curved ventrally, lacked lower jaws and had an inflated heart cavity. At 5 dpf, they did not recover any of the abnormalities seen at 3 dpf. (E) Acetylated tubulin staining of 2-dpf embryos injected with 5 ng of rtn4a MO revealed pathfinding mistakes of the lateral line. (F) Proportion of embryos with aberrant pathfinding of the lateral line at 2 dpf after injection of a mixture of 2 ng (n = 45) or 5 ng (n = 75) of each isoform of rtn4a-MO (arrow).
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