a Five-generation pedigree of a consanguineous Pakistani family with ten affected individuals (black-filled symbols). STR and single-nucleotide polymorphism marker haplotypes of all analyzed individuals are shown below each symbol. Diseased haplotype is marked as filled black bar. b Cerebral MRI scans of patient V-14, presenting with microcephaly and slanting forehead. c DNA sequence of homozygous control, heterozygous carrier and homozygous patient with c.465 G > C (p.W155C) mutation in exon 5 of RRP7A.

a, b DAB staining depicting the expression of RRP7A in the human parietal cortex in low a and higher b magnification of the ventricular zone (VZ). Scale bars, 5 mm a and 0.5 mm b. c, d Higher magnification of zones #1 and #2 boxed in b. RRP7A reactivity is high in cilia (open arrows) c and in RGCs (closed arrows) d. Scale bars, 40 µm. e IFM analysis of the region depicted in b showing expression of RRP7A (red) in RGCs marked with Vimentin (green) and localization to cilia (open arrows). Scale bar, 50 µm. Insert: shifted overlay of region boxed in the merged panel showing RRP7A expression in RGCs (closed arrow). Insert scale bar, 10 µm. f DAB staining depicting the expression of RRP7A in RGCs in a section of the temporal cortex and hippocampal formation. Scale bar, 5 mm. g, h Higher magnifications of the zone boxed #1 in f showing expression of RRP7A at the cortical plate (CP). Scale bar, 0.2 mm. h Higher magnification of the zone boxed in g showing expression of RRP7A in CP RGCs (closed arrows). Scale bar, 50 µm. i Higher magnification of the zone boxed #2 in f showing expression of RRP7A (left panel) in RGCs at the outer surface along the hippocampus (F: Fimbria). RGCs (closed arrow) in this region are further marked with Glial Fibrillary Acidic Protein (GFAP) (right panel). Scale bars, 0.2 mm. Low magnification with GFAP staining is also shown in Supplementary Fig. 2a. j IFM analysis of the area of fimbriae shown in (i) presenting expression of RRP7A (red) in RGCs marked with Vimentin (green). Scale bar, 0.1 mm. k Higher magnifications of the zone boxed in j showing expression of RRP7A in RGCs (closed arrow). Scale bar, 25 µm. IZ/OFL Intermediate zone/outer fiber layer.

a WB analysis of RRP7A expression in CRISPR clones of P19CL6 mouse stem cells holding WT RRP7A (P19CL6^Rrp7aWT#1 and P19CL6^Rrp7aWT#2) or mutated RRP7A; P19CL6^{Rrp7aΔ1/Δ18} (pArg59LysfsTer60/p.53-58del) and P19CL6^{Rrp7aΔ8/Δ33} (p.Arg48ProfsTer15/p.49-59del). b IFM analysis of CRISPR clone proliferation at day 3 post seeding in the absence of retinoic acid (RA). Nuclei were stained with DAPI (blue). Dotted lines outline cell colonies. Scale bars, 1 mm. c Quantification of proliferation of CRISPR clones measured by cell coverage (confluency) (data average of n = 3 independent experiments); Rrp7aWT#2 (P = 0.230), Rrp7aΔ1/Δ18 (P = 1.268E-05), Rrp7aΔ8/Δ33 (P = 5.372E-04)). d Quantification of relative levels of BrdU incorporation in WT and mutant clones (data average of n = 3 independent experiments; Rrp7aWT#2 (P = 0.617), Rrp7aΔ1/Δ18 (P = 0.004), Rrp7aΔ8/Δ33 (P = 5.635E-04)). e, f IFM analysis of the ability of CRISPR clones to undergo neurogenesis after 6 d and 9 e days of RA stimulation. The stippled lines in the upper panels indicate the lining of cellular clusters from where neurons are formed in WT clones. Neurons are marked with anti-ßIII-tubulin (upper panels: white; lower panels: green) and anti-MAP2 (lower panels: red), and nuclei are stained with DAPI (blue). Lower panels are shown as shifted overlays between ßIII-tubulin and MAP2. Scale bar upper panels, 0.1 mm and lower panels, 40 µm. g Quantification of relative levels of βIII-tubulin fluorescence in WT and mutant clones shown in f (data average of n = 3 independent experiments; Rrp7aΔ1/Δ18 (P = 6.244E-05), Rrp7aΔ8/Δ33 (P = 2.479E-04)). h WB analysis of WT clone P19CL6^Rrp7aWT#1 and mutant clone P19CL6^{Rrp7aΔ8/Δ33} at day 3, 6, and 9 of RA stimulation; antibodies as indicated. i–k Quantification of relative levels of PAX6 (day 3: P = 0.532; day 6: P = 2.696E-05; day 9: P = 0.025) (i), ßIII-tubulin (day 3: P = 0.232; day 6: P = 0.003; day 9: P = 2.400E-05) (j) and MAP2 (day 3: P = 0.774; day 6: P = 0.085; day 9: P = 1.117E-07) k shown in h (data average of n = 3 independent experiments). Data are represented as mean ± SD and significance was determined using an unpaired, two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s.: not significant.

a IFM analysis on the localization of RRP7A (green) to nucleoli (open arrows, light microscopy (LM)), the primary cilium (closed arrows, ARL13B, red) in serum-depleted cultures of control human dermal fibroblasts (HDF). The nucleus (nu) is stained with DAPI (blue). Scale bar, 20 µm. Insert scale bar, 5 µm. b Zoomed image of the cilium-centrosome axis depicted in a with the ciliary base/centrosome marked with anti-Pericentrin-2 (asterisk, PCTN-2, magenta). Scale bar, 5 µm. c IFM analysis of localization of RRP7A (red) to nucleoli (open arrows) in serum-supplemented cultures. Scale bar, 20 µm. Scale bar in zoomed image, 5 µm. d IFM analysis on the localization of RRP7A (green) to nucleoli (stippled-lined areas) and the primary cilium (ARL13B, red) in serum-depleted cultures of control HDFs (left panel) and patient HDFs (right panel). Scale bars, 10 µm. The insert shows magnification of boxed areas with shifted overlays between RRP7A (green, closed arrow) and ARL13B (red, closed arrow). The nucleus (nu) is stained with DAPI. Scale bars, 2 µm. e Quantification of the relative levels of RRP7A in nucleoli in control and patient HDFs shown in d (control = 151 cells; patient = 125 cells, data average of n = 5 independent experiments; P = 0.0004). f Quantification of the relative levels of RRP7A at the cilium-centrosome axis in control and patient HDFs shown in d (control = 84, patient = 91, data average of n = 4 independent experiments;; P = 0.762). Data are represented as mean ± SD and significance was determined using an unpaired, two-tailed Student’s t test. ***P < 0.001, n.s.: not significant.

a Left: representative northern blots of parallel gel runs of total RNA samples from control and patient HDFs with mutations in RRP7A (patient) and WDR62 (MCPH2 pt.), respectively. Black arrows indicate processing intermediates inferred from the analyses and gray arrows mark the migration of mature rRNA species as inferred from reprobing of the filters with probes targeting these RNA species. a Right: quantification of northern blots as log2 fold-change normalized to 45S and the control sample (data average of n = 3 independent experiments; 41S: P = 0.0004, 30S: P = 0.017, 21S: P = 0.027). Hybridization against 45S, and mature 18S and 28S rRNA were used as internal molecular markers. b Immunoprecipitation (IP) with anti-GFP was performed on lysates of cells transfected with constructs expressing either GFP (GFP), FLAG-RRP7A-GFP^WT (WT) or FLAG-RRP7A-GFP^W155C (MUT), and IPs were analyzed by WB using antibodies against NOL6 and NOL12. The experiments were repeated three times, and results from one representative experiment are shown. c IFM analysis on the localization of NOL6 and NOL12 (green) and RRP7A (red) to nucleoli (open arrows, differential interference contrast microscopy, DIC) in cultures of control and patient HDFs. The circumference of the nucleus is marked with a stippled line. Scale bar, 10 µm. d Quantification of the relative levels of NOL6 (ctr = 70 cells, pt = 66 cells, data average of n = 3 independent experiments; P = 5.749E-06) and NOL12 (ctr = 70 cells, pt = 73 cells, data average of n = 3 independent experiments; P = 0.808) in nucleoli in control and patient HDFs shown in c. Data are represented as mean ± SD and significance was determined using an unpaired, two-tailed Student’s t test. *P < 0.05, **P < 0.01, ****P < 0.0001, n.s.: not significant.

a IFM analysis of primary cilia in control and patient dermal fibroblasts (HDFs) cultured in the presence of serum. Primary cilia (arrows) are marked with anti-ARL13B (green) and anti-acetylated α-tubulin (Ac-tub, red), and nuclei are stained with DAPI (blue). Scale bars, 40 µm. Scale bar in zoomed image, 2 µm. b Quantification of ciliary frequency in control and patient HDFs serum depleted for 0–72 h (control ≥ 132 cells per time point, patient ≥125 cells per time point, data average of n = 3 independent experiments; 0 h: P = 0.002, 24 h: P = 0.268, 48 h: P = 0.349, 72 h: P = 0.397). c Quantification of ciliary length in control and patient HDFs serum depleted for 0–72 h (control ≥ 63 cells per time point, patient ≥ 82 cells per time point, data average of n = 4 independent experiments; 0 h: P = 0.507, 24 h: P = 0.053, 48 h: P = 0.440, 72 h: P = 0.169). d Overview of ciliary lengths in control HDFs (upper panels) and patient HDFs (lower panels) subjected to serum depleted for 48 h followed by serum addition for 0–24 h. Cilia are marked with anti-ARL13B (arrows, red) and centrosomes (ciliary basal bodies) are marked with anti-PCTN-2 (asterisks, white). Scale bar, 2 µm. e Quantification of cilia frequency based on data presented in d (control ≥ 131 cells per time point, patient ≥ 142 cells per time point, data average of n = 3 independent experiments; 6 h: P = 0.480, 12 h: P = 0.176, 16 h: P = 0.070, 20 h: P = 0.039, 24 h: P = 0.002). f Quantification of cilia length based on data presented in d (control ≥ 60 cells per time point; patient ≥ 60 cells per time point, data average of n = 3 independent experiments; 6 h: P = 0.479, 12 h: P = 0.908, 16 h: P = 0.444, 20 h: P = 0.001, 24 h: P = 0.0004)). Data are represented as mean ± SD and significance was determined using an unpaired, two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, n.s.: not significant.

a WB analysis of the level of RRP7A expression and phosphorylation of retinoblastoma protein (p-RB) and CDK1 (p-CDK1^T161) in control and patient HDF cells subjected to serum depleted for 48 h followed by serum addition for 0–24 h. b Quantification of relative levels of p-RB shown in a (data average of n = 4 independent experiments; 0 h: P = 0.537, 6 h: P = 0.105, 12 h: P = 0.016, 16 h: P = 0.023, 20 h: P = 0.005, 24 h: P = 5.035E-07). c Quantification of relative levels of p-CDK1^T161 shown in a (data average of n = 4 independent experiments; 0 h: P = 0.984, 6 h: P = 0.157, 12 h: P = 0.192, 16 h: P = 0.014, 20 h: P = 0.004, 24 h: P = 6.358E-08). d Quantification of relative levels of BrdU incorporation in growth-arrested control and patient HDFs stimulated with serum for 18 h (data average of n = 3 independent experiments; P = 0.001). Data are represented as mean ± SD and significance was determined using an unpaired, two-tailed Student’s t test. *P < 0.05, **P < 0.01, ****P < 0.0001, n.s.: not significant.

a Expression of rrp7a in 2 dpf zebrafish analyzed by in situ hybridization. Scale bars, 0.1 mm. b Bright-field images showing the morphology of three dpf rrp7^+/+ and rrp7a^−/− mutant zebrafish larvae. Scale bars, 0.2 mm. c–f Quantification of morphological differences between rrp7^+/+ and rrp7a^−/− zebrafish larvae. Trunk length c (P = 0.330), normalized eye diameter d (P = 4.563E-16), dorsal head size e (P = 4.243E-10) and lateral head size f (P = 4.139E-14) were quantified (n = 19, biologically independent animals). Measured lengths and areas are indicated in red lines in b (lower panel). g Haematoxylin and eosin (HE) staining of transverse tissue sections from a 3 dpf rrp7^+/+ (upper panels; i–iii) and rrp7a^−/− mutant (lower panels; i–iii) zebrafish larvae. Location of sections is indicated by red dotted lines in b (upper panel). Note the reduction in cells in the pallium (p) of rrp7a^−/− compared with rrp7^+/+ (marked in zoomed box, and quantified at right, n = 3, biologically independent animals). Data are presented as mean ± SD (P = 0.007). h hypothalamus, le lens, mc Meckel´s cartilage, mo medulla oblongata, ot optic tectum, sp subpallum. Scale bars, 0.1 mm. h mRNA rescue of rrp7a mutants. Normalized eye diameter and lateral head size were quantified and plotted as box and whiskers, whiskers min to max. i Left: northern blot (top) of total RNA from pooled zebrafish larvae analyzed with 18S rRNA and 28S rRNA probes, respectively, and qRT-PCR (bottom) displaying relative 18S/28S rRNA ratios with wt set to 1 (data average of n = 5 independent experiments). Data are presented as mean ± SD, no in cross phenotype: P = 0.296, in cross phenotype: P = 1.423E-04. Right: Northern blot analysis of processing intermediates of somatic (late) rRNA in rrp7a^+/− crosses and a schematic drawing of somatic rRNA processing intermediates inferred from northern blots. Hybridization against 45S, and mature 18S and 28S rRNA were used as internal molecular markers. Unpaired, two-tailed Student’s t test. (c–g and i) and one-way ANOVA (h, normalized head size: box 1 vs 3 P = 0.035, 2 vs 4 P = 0.050). **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s.: not significant.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.