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Additional Supporting Information may be found in the online version of this article.

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DVDY_22187_sm_suppfig1.tif228KSupp. Fig. S1. Expression of irxl1 in the trunk region was observed by whole-mount in situ hybridization at (left panels, 48–72 hours postfertilization [hpf]) and confirmed by reverse transcriptase-polymerase chain reaction (right panels, 24–72 hpf). The tissue used for RNA extraction is indicated by the boxes on the left images.
DVDY_22187_sm_suppfig2.tif182KSupp. Fig. S2. Splicing of irxl1 is disrupted in MOII morphants. A: Locations of the irxl1 morpholino oligos (MO). MOI is complementary to 25 bp spanning the translation initiation site of irxl1 mRNA (−17 to +8), MOII is complementary to 25 bp spanning the boundary of intron 3 and exon 4. B: Reverse transcriptase-polymerase chain reaction analysis of RNA isolated from wild-type and MOII-injected embryos at 25 hpf using primers a, b, c as indicated in (A). Primers a+b will amplify the correctly-spliced transcript (381 bp) whereas primers a+c will amplify a transcript with intron 3 unspliced (predicted size 344 bp). The correctly-spliced product (381 bp) is significantly reduced in MOII morphant, while the unspliced product (344 bp) is present in MOII morphant but not wild-type embryos, indicating that splicing of irxl1 is disrupted in MOII morphants.
DVDY_22187_sm_suppfig3.tif630KSupp. Fig. S3. The neuronal cell death phenotype is p53 dependent. A–F: Acridine orange staining of wild-type embryos (A,B), embryos injected with irxl1 morpholino oligonucleotides (MO; C,D) and embryos injected with both irxl1 MOI and p53 MO (E,F) at 25 hours postfertilization (hpf). G: Semiquantitative reverse transcriptase-polymerase chain reaction analysis of Δ113p53 and p21 expression in wild-type, irxl1 MOI morphant, irxl1 and p53 MO morphant, and control (Con) embryos at 25 hpf. Scale bar = 300 μm in A,C,E, 100 μm in B,D,F.
DVDY_22187_sm_suppfig4.tif1360KSupp. Fig. S4. Characterization of the raised zebrafish Irxl1 antibody. A–D: The Irxl1 antibody can detect the zebrafish Irxl1 fusion protein in a mouse myoblast cell line (C2C12). Twenty-four hours after transfection of pEGFP (A,B) or pIrxl1a-EGFP fusion genes (C,D) into C2C12 cells, the expression was detected by observing enhanced green fluorescent protein (EGFP) fluorescence under a fluorescence microscope (A,C) or by immunohistochemical staining using the Irxl1 Ab (B,D). Note that the expression of Irxl1-EGFP fusion protein was restricted to the nucleus. E: The Irxl1 antibody fails to detect the endogenous protein in zebrafish embryos and adult tissues by Western blotting. 40 μg of protein extracts from C2C12 cells transfected with pEGFP or pCS2-Irxl1a, 24 hours postfertilization (hpf)-, 48 hpf embryos, adult fish brain and muscle tissues were used in Western blotting by the Irxl1 Ab. The antibody detects the exogenously expressed but not endogenous Irxl1 protein (predicted size ∼ 40 kDa). P, a recombinant His-tagged Irxl1 protein used as a positive control. Detection of actin was used as a loading control.
DVDY_22187_sm_supptable1-2.docx18KSupporting Tables 1 and 2

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