Subcellular localization and Y2H analyses of wild type and <i>fan</i> mutant Wdr43.

<p>(A1–A4) IF images of HeLa cells immunostained with anti-WDR43 (green) and anti-B23 (red) antibodies followed by DAPI stain (blue) to visualize nuclear DNA. IF images of Hela cells transfected with EGFP tagged zebrafish wild type Wdr43 (B1–B4) or <i>fan</i> mutant Wdr43 (C1–C4). Anti-GFP antibody was used to increase the fluorescent signal of EGFP-tagged wild type and <i>fan</i> mutant Wdr43 expressed proteins. Stained cells were counterstained with anti-B23 (red) and DAPI (blue). (D) Y2H analysis of zebrafish full length (FL) Wdr43, truncated <i>fan</i> mutant Wdr43 (N), and Wdr43 C-terminal domain (C) interactions with zebrafish Utp4 and Utp15 proteins.</p

Subcellular localization of nucleolar proteins in WDR43 depleted HeLa cells.

<p>IF analysis of nucleolar protein localization in control siRNA (A, C, E, G) and <i>WDR43</i> (B, D, F, H) siRNA treated HeLa cells. WDR43 depleted cells exhibited mislocalized expression of TCOF1 (B), Mpp10 (D), Nucleolin (F) and Fibrillarin (H) (arrows), as compared to their respective control GFP siRNA treated cells (see arrows). Scale bar = 10 µm.</p

<i>fan</i> encodes zebrafish Wdr43/Utp5.

<p>(A) Left side: Chromosomal position of the <i>fan</i> locus (left). Sequencing trace data of wild type and <i>fan</i> mutant alleles, with red arrow indicating a premature stop codon in <i>fan</i> mutants (right). Comparison of the conserved nature of the amino acid mutated in <i>fan</i> zebrafish (purple) (middle). Schematic of the Wdr43 protein domains and <i>fan</i> mutant predicted premature stop codon (bottom). (B–E) Live images of zebrafish treated with control MO (CMO) or <i>fan</i> MO at indicated developmental times. (F–I) Alcian blue stained wild type (F, H) and <i>fan</i> mutant (G, I) embryos injected at the single cell stage with GFP control (<i>ctr</i>) or wild type <i>wdr43</i> mRNA. (I) Injected <i>wdr43</i> mRNA rescued <i>fan</i> mutant cartilage formation. (J) Percentage of control and <i>wdr43</i> mRNA injected <i>fan</i> mutant clutches exhibiting wild type, intermediate and <i>fan</i> mutant pharyngeal arch cartilage formation. Numbers of injected embryos scored are indicated at the top of each bar.</p

Ribosome biogenesis defects in <i>fan</i> mutant zebrafish.

<p>(A) Northern blot analysis of rRNA isolated from 30 and 50 hpf wild type (W) and <i>fan</i> mutants (M) using an oligonucleotide probe against the 5′ETS region of zebrafish pre-rRNA. Pre-rRNAs (a) and (b) are indicated. Methylene blue (MB) staining of the mature 28S and 18S rRNAs was carried out as a loading control. Quantitation of the ration of a/b was performed using Image J. (B) Western blot of whole-cell extracts treated with <i>WDR43</i> or EGFP control siRNA as indicated. (C) qRT-PCR analyses of human <i>WDR43</i> gene expression level in non-treated (NT), GFP or <i>WDR43</i> siRNA treated HeLa cells normalized to ß -actin. (D, E) Subcellular localization of mCherry-tagged Utp15 (red) in control GFP (D) and <i>WDR43</i> (E) siRNA treated HeLa cells counter stained with DAPI (blue).</p

Developmental expression pattern of zebrafish <i>wdr43</i> mRNA.

<p>(A–L) Whole mount ISH images of sense and anti-sense <i>wdr43</i> probes in developmentally staged zebrafish embryos. (M) RT-PCR results of <i>wdr43</i> gene expression in wild type and <i>fan</i> mutant embryos at indicated times (hpf). <i>fan</i> mutant mRNA was detected at 48 and 72 hpf, as indicated by digestion with DdeI, a unique restriction site generated by the <i>fan</i> point mutation. (N–Q) Sectioned ISH revealed discrete <i>wdr43</i> mRNA expression in neurepithelium (ne), pharyngeal arch tissues (pa), and gut (g) (arrows). (N′, P′) Higher magnification images of boxed regions in N, P, respectively. Sense controls did not exhibit staining (O, Q).</p

TCOF1 expression in stable <i>WDR43</i> shRNA cell lines.

<p>(A–O) IF analysis of TCOF1 (red) localization in control shRNA (A–D, M) and human <i>WDR43</i> shRNA (E–L, N, O) stable HeLa cell lines. All cells were positive for shRNA expression (green) and DAPI stained nuclei (blue). (P) Western blot analyses of WDR43 expression in stable <i>WDR43</i> shRNA cell lines. (Q–R) Quantification of TCOF1 positive nucleoli per cell (Q), and percent cells with fused TCOF1 positive nucleoli (R), in control and <i>WDR43</i> shRNA stable lines. The number of nucleoli was increased (Q), and the percentage of fused nucleoli was reduced (R) in WDR43 depleted cells, as compared to control cells. Sample numbers: Panel Q - ctr 290, 2a1 136, sh6 195, sh8 172, sh9 172, sh10 108; Panel R – ctr 268, 2a1 115, sh6 181, sh8 163, sh9 171, sh10 108.</p

Phenotype of <i>fan</i> mutants.

<p>(A–C′) Live images of developmentally staged wild type (A–C) and <i>fan</i> mutant (A′–C′) zebrafish. Arrow in A′ indicates necrotic cells in the presumptive eye region. Bracket in B′ shows necrosis in neural and pharyngeal arch tissues. Arrow in C′ points to the distinct hydrocephaly in <i>fan</i> mutant hind brain ventricles, arrowhead indicates incomplete choroid fissure closure and craniofacial defects. (D–D′) Alcian blue stained pharyngeal arch cartilages in 4dpf wild type (D) and <i>fan</i> mutant (D′). (E–K′) Whole mount ISH images of wild type (E–K) and <i>fan</i> mutants (E′–K′) for neural crest markers at indicated developmental stage. Arrows in (E′–G′) and asterisks in (I′–K′) indicate reduced gene expression in <i>fan</i> mutant embryos. Interestingly, <i>fan</i> mutant embryos exhibit similar <i>crestin</i> expression in the trunk NCCs (asterisks in H, H′) but reduced expression in cranial NCCs. (L–L′) <i>Tg(fli1a:EGFP)/fan</i> mutants (L′) exhibit reduced GFP expression in the pharyngeal arch region as compared to wild type embryos (L).</p

Inhibition of p53 signaling can partially rescue <i>fan</i> mutant craniofacial defects.

<p>(A–B) Whole mount IHC analysis of p53 expression in wild type (A) and <i>fan</i> mutants (B). (C, D) qRT-PCR analysis of <i>d113p53</i> (C) and <i>mdm2</i> (D) gene in wild type and <i>fan</i> mutants at 24, 48 and 72 hpf. (E–G) Bright field images of live 3 dpf wild type (E), <i>fan</i> mutant (F), and <i>fan</i> mutant injected with p53MO (G). (H) Percent zebrafish exhibiting normal eye development in uninjected (Uninj), control MO (CMO), and p53MO injected embryos. (I–K) TUNEL stained and sectioned wild type (WT) (I), <i>fan</i> mutant (J), and <i>fan</i> mutant injected with p53 MO (K). Arrows indicate apoptotic cells. (L) Statistical analyses revealed significantly reduced apoptosis in <i>fan</i> mutants injected with p53MO. (P value<0.01).</p