Deficiency in origin licensing proteins dramatically impairs cilia formation.

<p>a–b) Control (C) or ORC1-deficient cells were induced to enter G0 by serum starvation for 24 or 48 hr and processed to identify cilia using anti-acetylated tubulin and anti-γ-tubulin antibodies to mark the entire cilia or the basal body, respectively. Lower panel shows that in ORC1-hTERT fibroblasts immunostaining with α-acetylated tubulin reveals extended perinuclear microtubular arrays around the centrosome in distinction to the ordered alignment in control cells and as reported for other cilia defective cells <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003360#pgen.1003360-Mill1" target="_blank">[56]</a>. c) Control (C) or ORC1-deficient hTERT cells were monitored for long term cilia formation as above after the indicated numbers of days of serum depletion. d) Origin licensing proteins were knocked down with siRNA in control hTERT cells, serum starved for 24 hrs then analysed for cilia formation as above. Although a marked defect is observed in cilia formation up to 48 h post serum starvation, cilia can form in around 50% of the cells when examined 4–5 days post serum starvation. e) ORC1-P4 hTERT cells were transfected with empty plasmid or plasmid expressing GFP-tagged <i>ORC1</i> cDNA and positive cells detected with anti-GFP antibodies. The percent of GFP⁺ cells, representing those that have been successfully transfected, with cilia was assessed as in panel (d). <i>ORC1</i> cDNA expression resulted in rescue of the defect in cilia formation. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003360#pgen.1003360.s002" target="_blank">Figure S2a</a> shows cilia formation in a gfp⁺ versus gfp⁻ cells.</p

ORC1 Meier-Gorlin syndrome and IFT43 Sensenbrenner syndrome fibroblasts exhibit impaired chondroinduction.

<p>a–b) Phase contrast images (40×) of control (C) hTERT, ORC1-deficient MGS (ORC1-P4hTert) and <i>IFT43</i>-mutated Sensenbrenner (IFT43) patient derived fibroblasts at 0 hr and 24 hr following addition to aggrecan coated plates. Size distribution of aggregates from control (C), ORC1 and IFT43 fibroblasts following 24 hr micromass culture in aggrecan coated plates (n = 350 aggregates scored per line). Larger aggregate size was a feature of control fibroblasts following chondroinduction compared to ORC1 and IFT43 cells. c–d) Semi-quantitative RT-PCR amplification of <i>VEGFA</i> isoform a (upper band) and isoform c (lower band) either uninduced (Und) or during chondroinduction. Both isoforms were induced in control fibroblasts (C) upon chondroinduction. Whilst IFT43 cells exhibited higher endogenous levels of <i>VEGFA</i> isoform c, it was not maintained upon chondroinduction. Isoform a also was not induced after chrondroinduction. Similar findings were observed for ORC1 cells, although the high endogenous level of isoform c reduced less dramatically than that in IFT43 cells but did not increase in as in control cells. <i>ELP4</i> was used as an amplification control. Panel (d) shows the combined quantification for isoforms a and c from the above panel. Similar findings have been observed in three independent experiments. e) Type I collagen represents a negative marker for chondroinduction as its levels decrease as differentiated chondrocytes secrete a specific extracellular matrix. Consistent with this, <i>COL1A1</i> levels, as monitored by quantitative RT-PCR were found to decrease in control fibroblasts (C) during chondroinduction. Interestingly, both ORC1 and IFT43 defective patient derived cells exhibited similar levels of endogenous <i>COL1A1</i> compared to control but by 48 h, the levels had less dramatically diminished compared to control cells. The results represent the mean of three experiments. f–g) Analysis of a control hTERT cell line treated with control siRNA oligonucleotides (siC) or with oligonucleotides specific (si) for <i>ORC1, 4, 6, CDC6</i> or <i>CDT1</i>. Cells were uninduced (Und) or induced on a chondrogenic matrix then assayed for <i>VEGFA</i> expression as detailed in (c–d). Panel (g) shows the combined quantification for isoforms a and c from the above panel. Similar findings have been observed in two independent experiments.</p

Meier-Gorlin syndrome patients LBLs display impaired origin licensing capacity; some but not all lines show impaired S phase progression.

<p>(a) EBV uses virally encoded EBNA-1, oriP and the host cell origin licensing complex for replication. ORC activity was assessed by the replicative capacity of plasmid-294, which encodes OriP and EBNA-1 in a control LBL (C) and LBLs derived from MGS patients with mutations in <i>ORC1, ORC4, ORC6, CDC6</i> and <i>CDT1</i>. Following transfection of the EBV episome into LBLs and incubation to allow replication, episomal DNA was extracted and examined after <i>BamH1</i> or <i>BamH1+Dpn1</i> digestion using plasmid-294 as a probe. <i>Dpn1</i> degrades unreplicated plasmids that retain bacterial Dam-dependent methylation. The EBV episome has a single <i>BamH1</i> site causing linearization after digestion. Although replication of EBV is less efficient in LBLs compared to hTERT immortalised fibroblasts, ∼5% of the EBV plasmids underwent replication in control cells as shown by the presence of full length episomes (band 1) after <i>Dpn1+BamH1</i> digestion. The level in MGS patient LBLs is substantially reduced. For quantification, the level of the full length plasmid band (1) was plotted relative to one of the <i>Dpn1</i> digestion products (2) and normalised to that obtained in the control (C). Efficient episomal transfection was shown by the similar level of digestion products in all samples. Results represent the mean of two experiments. The reduction was highly significant (t-test, 1-tailed equal variance. Nomenclature used throughout: no significance (ns) P>0.05, * P<0.05, ** P<0.01). (b) Control (C) and ORC1 LBLs were BrdU labelled for 30 min and incubated for the indicated times before fluorescence-activated cell sorting (FACS). The percentage of early S phase cells was assessed. The rate of loss of BrdU⁺ early S phase cells represents the speed of S-phase progression. LBLs with mutations in <i>ORC1, ORC4</i> and <i>ORC6</i> show an impaired rate of S phase progression; CDT1-deficient LBLs were similar to control LBLs and the CDC6-deficient LBLs progressed more rapidly through S phase.</p

Deficiency in origin licensing proteins results in increased centrosome and centriole copy number.

<p>(a–b) Exponentially growing cells were stained with anti-γ-tubulin and anti-Centrin2 to allow visualisation of centrosomes and centrioles, respectively. Cells with >2 centrosomes or >4 centrioles were scored (b). Note that previous studies with ATR-SS cells were carried out using nocodozole to accumulate M phase cells but this analysis was carried out without nocodozole addition to avoid any impact of this drug on spindle assembly¹⁷. The inset picture (a) shows the types of abnormalities observed. I: normal G2 phase centrosomes and centrioles in control hTERT fibroblasts. ORC1 deficient (ORC1 P4 hTERT) fibroblasts have defects that include II: supernumerary centrosomes and centrioles, III: highly multiple centrioles, IV: centrioles distal from the centrosome. Control-hTERT-immortalised fibroblasts were subjected to ORC1 siRNA and analyzed as above. Analysis was also undertaken in ORC1-deficient hTERT fibroblasts and in ORC1-hTERT fibroblasts following transfection with <i>ORC1</i> cDNA. Similar findings were observed using a distinct antibody to mark centrioles (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003360#pgen.1003360.s001" target="_blank">Figure S1</a>). (c–d) Control fibroblasts were treated with the indicated siRNA and examined as in (b) and by Western Blotting to measure knockdown efficiency using the indicated antibodies.</p

Deficiency in origin licensing proteins impairs cilia function in response to platelet-derived growth factor (PDGF).

<p>a–b) Fibroblasts were induced to enter G0 phase following serum depletion for 48 hr. PDGF-AA or –BB and BrdU was then added and the percentage of S phase cells, monitored as BrdU⁺ cells, was estimated by immunofluorescence 11 (a) and 24 hr (b) later. The receptor for PDGF-AA is located in cilia whilst the –BB receptor is on the cell membrane. c) Analysis as in a) following the indicated siRNA. d) Cellular localisation of PDGFR-α or β. Anti-PDGFR-α or –β antibodies were used to examine the localisation of the two PDGF isoforms in control (C) or ORC1-P4 hTERT cells. PDGFR-α localised to the cilia, identified using anti-acetylated tubulin in control and ORC1-P4 cells although fewer cilia formed in the latter cells. PDGFR-β showed pan cellular localisation but did not co-localise with the cilium. e) Cells were induced to enter G0 phase following serum depletion for 48 hr. Serum was then re-added and the fraction of BrdU⁺ S phase cells monitored at the indicated times. The top panel shows the results with a control (C) primary fibroblasts, 48BR, primary fibroblasts from Sensenbrenner syndrome patients (<i>IFT43</i>-mutated and <i>WDR35</i>-mutated), PCNT defective fibroblasts and an ORC1 deficient line MGS cells. Both Sensenbrenner syndrome lines and PCNT cells showed a delayed S phase entry, similar to ORC1 defective MGS, compared to the control primary line. f) Analysis of a control hTERT immortalised cell line either without knockdown (C), treatment with control siRNA oligonucleotides (siC) or with oligonucleotides specific (si) for <i>ORC1, ORC4, ORC6, CDC6</i> or <i>CDT1</i>. Knockdown efficiency was assessed and was similar to that observed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003360#pgen-1003360-g002" target="_blank">Figure 2</a>. Note that the control hTERT immortalised line (C) enters S phase more rapidly that the primary fibroblasts line making it difficult to allow a direct comparison between the S phase entry kinetic defects observed in the Sensenbrenner syndrome primary lines in (e).</p

Wnt5a Deficiency Leads to Anomalies in Ureteric Tree Development, Tubular Epithelial Cell Organization and Basement Membrane Integrity Pointing to a Role in Kidney Collecting Duct Patterning

<div><p>The Wnts can be considered as candidates for the Congenital Anomaly of Kidney and Urinary Tract, CAKUT diseases since they take part in the control of kidney organogenesis. Of them <i>Wnt5a</i> is expressed in ureteric bud (UB) and its deficiency leads to duplex collecting system (13/90) uni- or bilateral kidney agenesis (10/90), hypoplasia with altered pattern of ureteric tree organization (42/90) and lobularization defects with partly fused ureter trunks (25/90) unlike in controls. The UB had also notably less tips due to <i>Wnt5a</i> deficiency being at E15.5 306 and at E16.5 765 corresponding to 428 and 1022 in control (p<0.02; p<0.03) respectively. These changes due to <i>Wnt5a</i> knock out associated with anomalies in the ultrastructure of the UB daughter epithelial cells. The basement membrane (BM) was malformed so that the BM thickness increased from 46.3 nm to 71.2 nm (p<0.01) at E16.5 in the <i>Wnt5a</i> knock out when compared to control. Expression of a panel of BM components such as <i>laminin</i> and of <i>type IV collagen</i> was also reduced due to the <i>Wnt5a</i> knock out. The <i>P4ha1</i> gene that encodes a catalytic subunit of collagen prolyl 4-hydroxylase I (C-P4H-I) in collagen synthesis expression and the overall C-P4H enzyme activity were elevated by around 26% due to impairment in <i>Wnt5a</i> function from control. The compound <i>Wnt5a</i>^+/-;<i>P4ha1</i>^+/- embryos demonstrated <i>Wnt5a</i>^-/- related defects, for example local hyperplasia in the UB tree. A R260H WNT5A variant was identified from renal human disease cohort. Functional studies of the consequence of the corresponding mouse variant in comparison to normal ligand reduced Wnt5a-signalling <i>in vitro</i>. Together Wnt5a has a novel function in kidney organogenesis by contributing to patterning of UB derived collecting duct development contributing putatively to congenital disease.</p></div

<i>Wnt5a</i> deficiency associates to compromised basement membrane integrity and integrin <i>Itga1</i> expression.

<p>Wild-type (Wt) and <i>Wnt5a</i>^-/- kidneys at E11.5 and E16.5 were prepared and subjected to electron microscopic (EM) inspection (A-F). The noted phenotypes were quantified by morphometric (G-H). The basement membrane (BM) that forms normally an extracellular matrix (ECM) sheet between ureteric bud derived epithelial tree and adjacent mesenchyme (A, D) becomes severely compromised in the <i>Wnt5a</i> deficient embryonic kidney already at the initiation of organogenesis at E11.5 (compare B, C with A, arrows) and is prominent also at E16.5 (compare E, F with D, arrows). Please note that the BM of the <i>Wnt5a</i> deficient kidney loses it integrity and the BM is more extended when compared to control BM (compare B, C, E, F with A, D, arrows). G, H) Quantitation of the BM (A-F) in depicting that the width of the BM and the distance of the BM from the cell membrane is increased in the absence of <i>Wnt5a</i> function when compared to control. Data are presented as means ± SD. *p < 0.05, n = 4 mice/group. Scale bars, A-F 200 nm.</p

<i>Wnt5a</i> deficiency increases the collagen prolyl 4-hydroxylase I (P4ha1) expression and the compound +/- is characterized by severe kidney filter anomaly.

<p>The kidneys were prepared and subjected to <i>collagen prolyl 4-hydroxylase I (P4ha1)</i> gene expression (A), total Collagen P4H (C-P4H) enzyme activity assays (B) or electron microscopic inspection (C-J) at E16.5 or in the adult. <i>Wnt5a</i> deficiency has increased the <i>P4ha1</i> gene expression (A) and C-P4H enzyme activity (B) when compared to control. The ureteric bud derived collecting duct basement membrane (BM) remains normal at E16.5 in the <i>Wnt5a+/-</i> and <i>P4ha1+/-</i> (compare D, E with C) but the BM is malformed in the <i>P4ha1+/-; Wnt5a+/-</i> at E16.5 (compare F with C, D, E). However, the kidney filter, the glomerular BM and the foot processes are compromised in the kidney of the <i>P4ha1+/-</i> and the <i>Wnt5+/-</i> mice at the age of seven months (compare H, I with G) and this is even more severe in the kidneys derived from the <i>P4ha1+/-; Wnt5a+/-</i> compound heterozygous mice (compare J with G, H, I). GBM; glomerular BM, P, podocyte, E, endothelial cell. The data in A and B are shown as means ± SD, n = 4. Scale bars, C-J 1μm.</p

<i>Wnt5a</i> deficiency leads to severe metanephric kidney anomalies.

<p>Embryonic urogenital systems (UGS) (A-D) and their kidneys (E-L) were dissected at E15.5 and E16.5. The UGS were inspected either as unstained (A-D) or their kidneys we subjected to Troma-1 antibody staining as whole mount and analysed with the optical projection tomography (OPT) to identify the pattern of the ureteric bud (UB) tree and the terminal UB derived tree tip counts (M) or sectioned (I-L). A) A normal urogenital system. The kidney (K), the gonad (G) and the adrenal gland (A) are marked. The <i>Wnt5a</i> deficiency leads to three categories of phenotypes; B) a kidney with duplex UB highlighted in the boxed image (the stars in B´), kidney hypoplasia (compare B with A), bilateral (C) or unilateral (D) renal failure. The OPT reveals variation noted in the pattern of UB tree development in the <i>Wnt5a</i> deficient embryonic kidneys when compared to control (compare F—H with E). The altered UB tree pattern can be depicted in the sections of the <i>Wnt5a</i> deficient kidneys when compared to control (compare J with I, dotted line, arrows). <i>Wnt5a</i> deficiency enlarges also the Bowman´s capsule lumen (asterisk) from control one (compare L with K, stars). Counting of the UB terminal tips from the OPT data indicates reduction in their number due to <i>Wnt5a</i> deficiency from controls M). Data in M are shown as means ± SD, <i>n</i> = 4–5 kidneys/group. Scale bars, A-D 400 μm, E-H 200 μm and I-L 50 μm. * <i>P</i> <0.05.</p

A human WNT5A R260H variant from a CAKUT cohort reduces signalling possibly via the frizzled receptor binding pocket widening as judged by simulation.

<p>The putative influence of the WNT5A R260H variant discovered in human CAKUT disease cohort was modelled by simulation by using the xWnt8 structure as the reference. A) The R260H variant in the mouse Wnt5a-GFP reduces the capacity of the Wnt5a to inhibit the Wnt3a induced <i>Top Flash</i> luciferase activity when compared to the potential of the native Wnt5a by 17% (p<0.05). B) The simulation suggests that the human R260H variant broadens the WNT5A frizzled receptor binding pocket from 3.06 nm (Wt, in red) to 3.74 nm (R260H WNT5A, in pale blue circle and dotted line). The spatial location of the residue 260 variant in the patient with CAKUT is highlighted as a green circle. CM, conditioned media. The data are shown as means ± SD, n = 7/group, * <i>p</i> < 0.05.</p