Figure 1.
hkc is defective in thymus development.
(A) Whole-mount in situ hybridization of 6-dpf wild-type (WT, top) and hkc (bottom) embryos using rag1 (left), ikaros (middle), and tcrβ (right) probes. Ventral views are shown. Embryos were treated with H2O2 to bleach pigment cells in the rag1 group. (B) Quantitative PCR analysis of indicated genes in whole bodies of 7-dpf WT (open bars) and hkc (closed bars) embryos. Expression in WT embryos was normalized to 1. Results represent averages and standard errors of four independent measurements. Asterisks, p<0.05. (C) Whole-mount in situ hybridization of WT (top) and hkc (bottom) embryos at stage 21 using ikaros (left) and gata1 (center) probes. Dorsal views of posterior regions are shown. Right panels show Giemsa-May-Grunwald-stained red blood cells of 7 dpf WT (top) and hkc (bottom) embryos. (D) Transplantation of rag1-EGFP transgenic thymocytes into embryos. Top images show green fluorescence signals and red CMTMR signals in the thymus of WT (left) and hkc (right) recipients at 1 day after transplantation. Bottom plots indicate numbers of donor-derived EGFP+ cells in WT and hkc thymuses. Plots of left and right thymus in individual recipients are indicated. 15 and 11 recipients of WT and hkc, respectively, were analyzed. (E) Whole-mount in situ hybridization of WT (top) and hkc (bottom) embryos using dlx2 (stage 26), pax9 (6 dpf), and foxn1 (5 dpf) probes. Ventral views are shown. Embryos were treated with H2O2 to bleach pigment cells in the foxn1 group. Four panels on the right show Alcian blue staining of 9-dpf WT (top) and hkc (bottom) larvae. Ventral and lateral views are shown. Numbers indicate pharyngeal arches.
Figure 2.
A missense mutation in WDR55 is responsible for hkc phenotype.
(A) hkc was mapped on linkage group (LG) 18 and confined in the 23 kb region on scaffold 567. Recombination rates of 407 hkc embryos from hkc/+ (cab-derived)×kaga crosses and cM distances from hkc to neighboring markers are shown. These markers are located in a single BAC Md0218G12. Indicated two genes were predicted in the mapped region. (B) Sequences within the WDR55 coding region of WT (left) and hkc (right) genomic DNA and predicted amino acid sequences. (C) Predicted amino acid sequence of medaka (Ol) WDR55 was compared with zebrafish (Dr), mouse (Mm), and human (Hs) WDR55 sequences. Asterisks and dots indicate residues that are shared among all four species and three species, respectively. Arrows indicate WD repeat domains. Red box indicates glycine residue that is replaced with arginine in hkc mutants. (D) Predicted structure of medaka WDR55 protein. Filled boxes indicate WD repeat domains. Amino acid residues and predicted secondary structures of the second WD repeat domain are also shown. (E) 1-cell-stage embryos from +/hkc×+/hkc matings were injected with WT- or hkc-derived WDR55 mRNA and EGFP mRNA, and whole-mount in situ hybridization using rag1 probe was carried out at 6 dpf. P-values were calculated using κ-square test. (F) 50 µM of morpholino that blocks splicing of WDR55 (sp-MO) was injected into WT embryos. Three images appearing on top left show dorsal views of control embryos (cont.) and morphants (sp-MO) at stage 25. Four images appearing at the bottom show ventral views of control (cont.) and 5-dpf morphants (sp-MO) hybridized with rag1 probe. Numbers below images indicate the numbers of embryos showing phenotypes of the images over the numbers of total embryos examined. Top right images show ethidium bromide (EtBr)-stained gels of RT-PCR products for two WDR55 exons neighboring the position of splicing-inhibiting morpholino. Total RNAs from two individual control embryos (cont.) and two individual morphants (sp-MO) at stage 25 were examined. cDNA was synthesized in the absence (−) or presence (+) of reverse transcriptase. Adult WT cDNA and genomic DNA were also amplified. cDNA for cytoplasmic actin (CA) was amplified to verify the quality of cDNA synthesized. (G) 50 µM of morpholino that was designed to block translation of WDR55 mRNA was injected into WT embryos. Three images show ventral views of control (cont.) and 6-dpf morphants (ATG-MO) hybridized with rag1 probe. Numbers below images indicate the numbers of embryos showing phenotypes of the images over the numbers of total embryos examined.
Figure 3.
WDR55 modulates nucleolar rRNA production and cell cycle progression.
(A) Intracellular localization of transfected EGFP, EGFP-WDR55WT, and EGFP-WDR55hkc (green) along with co-transfected t-HcRed1-fibrillarin in 293T cells. Single fluorescence images and merged images are shown. Rightmost panels show images of single cells isolated from stage-19 embryos that were administered at 1-cell stage with mRNAs of indicated genes. (B) Localization of endogenous WDR55 in NIH3T3 cells identified by antibody staining. Single fluorescence images and merged images of WDR55 (green) and fibrillarin (red, top panels) or B23 (red, bottom panels) are shown. (C) Western blotting of whole cell lysates of control or WDR55-siRNA-transfected NIH3T3 cells at 44 hours after transfection using anti-calnexin or anti-WDR55 antibody. (D) Detection of rRNA processing intermediates by Northern blot hybridization with 5.8S rRNA probe of total RNA (0.5 µg per lane) isolated from untransfected, control-siRNA-transfected, and WDR55-siRNA-transfected NIH3T3 cells. Asterisks indicate accumulation of processing intermediates in NIH3T3 cells transfected with WDR55-siRNA. An image of methylene blue (MB) staining is also shown (left). The amount of housekeeping glyceraldehyde-3-phosphate dehydrogenase mRNA measured by quantitative RT-PCR was not significantly different among same-weight total cellular RNAs isolated from untransfected, control-siRNA-transfected, and WDR55-siRNA-transfected cells. Along with the data showing that the intensities of the bands for 28S, 18S, and 5.8S rRNAs were comparable among these three groups of cells, these results suggest that the total amounts of rRNAs were comparable among these three groups of cells. (E) Immunoprecipitation (IP) of U2OS cell lysates using anti-WDR12, anti-Pes1, and anti-Bop1 antibodies, or normal rat IgG. 2.5% of input lysate before IP was also electrophoresed. Western blot (WB) detection is shown for Pes1 (top) and WDR55 (bottom). (F) Quantitative RT-PCR analysis of indicated genes in control (open bars) and WDR55-siRNA-transfected (closed bars) NIH3T3 cells. Expression levels in control cells were normalized to 1. Results represent averages and standard errors of three independent measurements. Asterisks, p<0.05. (G) Cell cycle analysis of control (left) and WDR55-siRNA-transfected NIH3T3 cells by BrdU and 7-AAD staining. Numbers indicate frequency of cells in indicated squares. Lower left box, upper box, and lower right box show cells in G1 phase, S phase, and G2/M phase, respectively. Representative results of three independent experiments are shown.
Figure 4.
Defective rRNA processing and p53 activation in hkc mutants.
(A–E) Detection of rRNA processing intermediates by Northern blot hybridization. Total cellular RNAs (0.5 µg per lane for probes A, C, and E; 0.1 µg per lane for probes B and D) in 7-dpf embryos from WT, siblings (Sib), and hkc mutants were electrophoresed in agarose gels and was hybridized with indicated probes (panels A, B, and D). Images of methylene blue (MB) staining are also shown. Indicated amount of total cellular RNAs in 7-dpf embryos from WT, siblings (Sib), and hkc mutants were electrophoresed in polyacrylamide gel and hybridized with probe C (panel E). Predicted structures of intermediates a-c and positions of probes A–E are drawn (panel C). In panel E, the hkc lane shows an increase in slowly electrophoresed signal (designated as X), perhaps corresponding to the mixture of the intermediates described in panel C. The amount of housekeeping cytoplasmic actin mRNA measured by quantitative RT-PCR was not significantly different among same-weight total cellular RNAs isolated from WT, Sib, and hkc mutants. Along with data showing that the intensities of the bands for 28S, 18S, and 5.8S rRNAs were comparable among these three groups of cells, these results suggest that the total amounts of rRNAs were comparable among these three groups of the cells. (F) Quantitative RT-PCR analysis of indicated genes in whole bodies of 7-dpf WT (open bars) and hkc mutants (closed bars). Results represent averages and standard errors of four independent measurements. Asterisks, p<0.05. (G) Phenotypes of WDR55hkc/hkcp53Y186X/Y186X embryos. Whole-mount in situ hybridization of rag1 was carried out with 5- to 7-dpf embryos obtained from WDR55+/hkcp53Y186X/Y186X× WDR55+/hkcp53Y186X/Y186X crosses or WDR55+/hkcp53+/Y186X× WDR55+/hkcp53+/Y186X crosses. (H) Quantitative RT-PCR analysis of indicated genes in whole bodies of 7-dpf WT (open bars) and p53Y186X/Y186X mutants (closed bars). Results represent averages and standard errors of four independent measurements. Asterisks, p<0.05.
Figure 5.
How does hkc mutation result in defects in thymus development?
(A) Quantitative RT-PCR analysis of WDR55 in indicated organs of adult WT medaka. The expression in kidney was normalized to 1. Shown are averages and standard errors of three independent measurements. (B) Quantitative RT-PCR analysis of WDR55 in whole bodies of WT medaka at indicated stages. The expression at stage 39 was normalized to 1. Shown are averages and standard errors of four independent measurements. (C) Whole-mount in situ hybridization of WT embryos at stage 19 using WDR55 antisense (left) and sense (right) probes. Dorsal views (top) and lateral views (bottom) are shown. (D) Left ventrolateral views of WT (top) and hkc (bottom) embryos at stage 36 (left) and stage 38 (right). L, liver; G, gall bladder; S, spleen. (E) cDNAs from whole medaka embryos at indicated stages from the mating of kaga females with cab males were amplified for WDR55, followed by cab genotype-specific restriction digestion with BstNI. cDNAs from two individual embryos were analyzed for each stage. cDNAs from adult cab, kaga, and their heterozygotes are shown on the rightmost three lanes. M: size marker. (F) Cab/kaga heterozygous females and kaga males were mated and embryos were selected for individuals carrying kaga-derived WDR55 loci at both alleles. cDNAs from whole medaka embryos at indicated stages were amplified for WDR55, followed by cab genome-specific restriction digestion. (G) Stability of WDR55 proteins. 293T cells were transfected with EGFP (×), EGFP-WDR55WT (○), or pZsProSensor-1 (△) and treated with cycloheximide (CHX) for indicated periods. Fluorescence intensity was measured with a flow cytometer and relative mean fluorescence intensity was plotted. Shown are averages and standard errors of three independent measurements. (H) Detection of BrdU incorporation in WT (top) and hkc (bottom) embryos. Brown, BrdU; blue, hematoxylin. Thymuses of 7-dpf embryos (left) and eyes at stage 29 (middle panels and high-magnification images in right panels) are shown.
Figure 6.
Zebrafish WDR55 mutant shows defect in thymus development.
(A) Retroviral insertion in WDR55 locus of zebrafish hi2786B mutants. Uppercase letters indicate exons with predicted amino acids. Arrow indicates WDR domain. (B) Transcription of WDR55 3′ region and GAPDH in hi2786B (−/−) and heterozygous (+/−) zebrafish larvae at 6 dpf. cDNA was synthesized in the absence (−) or presence (+) of reverse transcriptase (RT). (C) Phenotypes of WT (top) and hi2786B (bottom) zebrafish. Far left panels show lateral views of 6-dpf larvae. Arrows indicate swim bladder. Second panels from the left show whole-mount in situ hybridization of 6-dpf larvae using rag1 probe. Ventral views are shown. Embryos were treated with H2O2 to bleach pigment cells. Middle panels show hematoxylin-eosin (HE) staining of transverse sections at 6 dpf. Broken blue lines indicate the thymus. Second panels from the right show Alcian blue staining of 6-dpf larvae. Ventral views are shown. Numbers indicate pharyngeal arches. Far right panels show HE staining of transverse sections of the eyes at 6 dpf.
Figure 7.
WDR55 deficiency in mouse causes developmental arrest before implantation.
(A) Genotype of mouse embryos from Wdr55+/−× Wdr55+/− crosses at indicated embryonic days. (B) Genotype of mouse progenies from the mating of Wdr55+/− females×Wdr55+/+ males (top) and Wdr55+/+ females×Wdr55+/− males (bottom). (C) Thymus development in Wdr55+/− embryos. Cryosections of developing thymus in Wdr55+/+ (left) and Wdr55+/− (right) embryos at E11.5 (top) and E15.5 (bottom) were stained with anti-CD45 (green) and anti-cytokeratin (red) antibodies.