ZFIN ID: ZDB-FIG-180202-2
Joris et al., 2017 - Number of inadvertent RNA targets for morpholino knockdown in Danio rerio is largely underestimated: evidence from the study of Ser/Arg-rich splicing factors. Nucleic acids research   45:9547-9557 Full text @ Nucleic Acids Res.
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Fish:
Knockdown Reagent:
Anatomical Terms:
Stage Range: Prim-5 to Protruding-mouth
PHENOTYPE:
Fish:
Observed In:
Stage Range: Prim-5 to Protruding-mouth

Fig. 2

srsf5a−/−and srsf5b−/− did not show any developmental defects, but presented an overexpression of several homologous SR genes. (A) Two TALEN pairs were designed to target exon 2 or exon 4 of the srsf5a locus. TALEN pairs 1 and 2 generated of a deletion of, respectively 11 (Δ11) and 5 nt (Δ5), resulting in the production of a protein truncated in the RRM1 domain. (B) Quantitative RT-PCR to measure mRNA expression of sr genes in wild-type (wt), srsf5a mutants, morphants and ctrlMO microinjected embryos at 24 hpf. A strong decrease of srsf5a mRNA levels was observed in mutants compared to wt, suggesting the loss of Srsf5a protein in the mutant. In contrast, an upregulation of srsf1b, srsf2b, srsf3a, srsf5b and srsf6a was found. No such differences were observed in morphants compared to injected control embryos or wt. The data represent mean ± S.D. expression relative to the ef1alpha reference gene of at least three independent experiments. One-way ANOVA followed by a Tukey's multiple comparison test was used for statistical analysis. *, **, ***Mutants are statistically different from wt (*P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001). (C) Fluorescent in situ hybridization using a pax6b probe followed by nuclear staining using draq7® in srsf5a−/− mutants and wt. No phenotype could be detected. (D) A CRISPr (Cr) was designed to target srsf5b exon2 and allowed us to obtain three different srsf5b mutants presenting 5, 11 or 14 bases deletion. The three mutations led to the production of a truncated protein in the RRM1 domain. (E) Comparison of SR genes expression level between srsf5b homozygous mutants (including the three mutant lines) and wt embryos at 24 hpf showed an overexpression of srsf2a, srsf3a and srsf3b. A drastic decrease of srsf5b expression confirmed its depletion in mutants. All data are expressed as the mean ± SEM. A one-way ANOVA was used for statistical analysis, followed by a multiple comparison Tukey's test. *, **, *** Mutants are statistically different from wt (*P ≤ 0.05, **P ≤ 0.01).

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
pax6b WT control Protruding-mouth retinal ganglion cell layer ISH
Protruding-mouth retinal inner nuclear layer ISH
srsf5aulg0030/ulg0030 control Protruding-mouth retinal ganglion cell layer ISH
Protruding-mouth retinal inner nuclear layer ISH
srsf1b WT + MO1-srsf5a control Prim-5 whole organism RTPCR
srsf5aulg0030/ulg0030 control Prim-5 whole organism RTPCR
srsf2a srsf5bulg0032/ulg0032 control Prim-5 whole organism RTPCR
srsf5bulg0033/ulg0033 control Prim-5 whole organism RTPCR
srsf2b WT + MO1-srsf5a control Prim-5 whole organism RTPCR
srsf5aulg0030/ulg0030 control Prim-5 whole organism RTPCR
srsf3a WT + MO1-srsf5a control Prim-5 whole organism RTPCR
srsf5aulg0030/ulg0030 control Prim-5 whole organism RTPCR
srsf5bulg0032/ulg0032 control Prim-5 whole organism RTPCR
srsf5bulg0033/ulg0033 control Prim-5 whole organism RTPCR
srsf3b srsf5bulg0032/ulg0032 control Prim-5 whole organism RTPCR
srsf5bulg0033/ulg0033 control Prim-5 whole organism RTPCR
srsf5a WT + MO1-srsf5a control Prim-5 whole organism RTPCR
srsf5aulg0030/ulg0030 control Prim-5 whole organism RTPCR
srsf5b WT + MO1-srsf5a control Prim-5 whole organism RTPCR
srsf5aulg0030/ulg0030 control Prim-5 whole organism RTPCR
srsf5bulg0032/ulg0032 control Prim-5 whole organism RTPCR
srsf5bulg0033/ulg0033 control Prim-5 whole organism RTPCR
srsf6a WT + MO1-srsf5a control Prim-5 whole organism RTPCR
srsf5aulg0030/ulg0030 control Prim-5 whole organism RTPCR
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
srsf5aulg0030/ulg0030 control Prim-5 whole organism srsf1b expression increased amount, abnormal
Prim-5 whole organism srsf2b expression increased amount, abnormal
Prim-5 whole organism srsf3a expression increased amount, abnormal
Prim-5 whole organism srsf5a expression decreased amount, abnormal
Prim-5 whole organism srsf5b expression increased amount, abnormal
Prim-5 whole organism srsf6a expression increased amount, abnormal
Protruding-mouth retina cell organized, normal
Protruding-mouth retinal ganglion cell layer organized, normal
Protruding-mouth retinal inner nuclear layer organized, normal
srsf5bulg0032/ulg0032 control Prim-5 whole organism srsf2a expression increased amount, abnormal
Prim-5 whole organism srsf3a expression increased amount, abnormal
Prim-5 whole organism srsf3b expression increased amount, abnormal
Prim-5 whole organism srsf5b expression decreased amount, abnormal
srsf5bulg0033/ulg0033 control Prim-5 whole organism srsf2a expression increased amount, abnormal
Prim-5 whole organism srsf3a expression increased amount, abnormal
Prim-5 whole organism srsf3b expression increased amount, abnormal
Prim-5 whole organism srsf5b expression decreased amount, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Nucleic acids research for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Nucleic Acids Res.